Methods and compositions relating to leptin antagonists

ABSTRACT

Devices, methods and compositions for treating cardiovascular disorders are configured for sustained-release of leptin antagonist into a blood vessel in order to down-regulate an expression or activity of leptin in a cardiovascular tissue that is affected by a disorder.

RELATED APPLICATIONS

The instant application claims priority as a continuation of U.S. patentapplication Ser. No. 15/000,062 filed Jan. 19, 2016, which is acontinuation in part under 35 U.S. C. 111(a) from Internationalapplication No. PCT/IL2015/050866 filed on 27 Aug. 2015, which takespriority from U.S. provisional patent application 62/188,676 filed 5Jul. 2015; U.S. Utility patent application Ser. No. 14/730,282 filed 4Jun. 2015; and U.S. provisional patent application 62/120,966 filed 26Feb. 2015, all four documents which are included by reference as iffully-set forth herein.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to the field of medicine, and in someembodiments to devices and compositions comprising a leptin antagonistformulated for localized release of a leptin antagonist at the site oftreatment as well as methods of using such compositions for treatingdisorders, including cardiovascular disorders.

Cardiovascular disease (CVD) is a class of diseases that involve theheart and/or the blood vessels. Several studies have relatedinflammatory markers to cardiovascular disease (CVD) and several assaysfor inflammatory markers are commercially available. For example,C-reactive protein (CRP), a common inflammatory marker, has been foundto be present in increased levels in patients who are at risk forcardiovascular disease [Karakas and Koenig, 2009 Herz 34 (8): 607-13]while osteoprotegerin, which is involved with regulation of NF-κB, hasbeen found to be a risk factor for cardiovascular disease and mortality[Venuraju et al., 2010 J. Am. Coll. Cardiol. 55 (19): 2049-61].

As a result of these findings, the number of inflammatory marker testsordered by clinicians for CVD risk prediction has grown rapidly.However, to date there is no consensus among professionals as to howthese markers of inflammation should be unsed as a basis for clinicaltreatment.

Although it has been shown that some cardiovascular disorders canbenefit from suppression of inflammation-related processes and cellularproliferation as part of a remodeling response (e.g. use of locallyreleased cytotoxic drugs such as paclitaxel or sirolimus in preventingrestenosis or use of doxycycline in treatment of abdominal aorticaneurysm (AAA)), to date, there is no evidence to suggest thatcardiovascular disease can benefit from anti-inflammatory treatment.

Leptin is a naturally occurring pleiotropic molecule that regulates foodintake as well as metabolic and endocrine functions. Leptin also plays aregulatory role in immunity, inflammation, and hematopoiesis.

The human leptin precursor is a linear polypeptide 167 amino acidresidues long represented by NCBI Reference Sequence NP_000221.1 (SEQ IDNO 1) encoded by the mRNA having the nucleotide sequence NCBI ReferenceSequence NM_000230. Residues 1-21 of the sequence constitute the signalpeptide while residues 22-167 constitute the mature hormone.

Leptin antagonists are also known, see for example, U.S. Pat. No.7,307,142 and U.S. Pat. No. 8,969,292.

SUMMARY OF THE INVENTION

The invention, in some embodiments, relates to the field of medicine,and more particularly to methods and devices that use leptinantagonists. In some embodiments, the invention relates to compositionscomprising a leptin antagonist formulated for localized release of aleptin antagonist at the site of treatment as well as methods of usingsuch compositions for treating disorders, including cardiovasculardisorders.

According to an aspect of some embodiments of the invention, there isprovided a method of treatment comprising: exposing in vivo tissue of asubject in need thereof to local administration of apharmaceutically-effective amount of leptin antagonist, therebyproviding a therapeutic effect to the tissue. In some embodiments, thetissue is substantially continuously exposed to apharmaceutically-effective amount of leptin antagonist for a period ofnot less than three days, not less than 5 days, not less than 8 days andeven not less than 14 days.

According to an aspect of some embodiments of the invention, there isalso provided a method of treatment comprising implanting in contactwith tissue in the body of a subject in need thereof a compositionconfigured for in vivo local administration of leptin antagonist,thereby providing a therapeutic effect to the tissue. In someembodiments, the configuration for the in vivo release is such that whenthe composition is implanted in vivo, leptin antagonist is released fromthe composition in a pharmaceutically-effective amount for a period ofnot less than three days, not less than 5 days, not less than 8 days andeven not less than 14 days.

In some embodiments of the methods, the need is that the subject suffersfrom at least one pathology selected from the group consisting of:cardiovascular disease; remodeling of stable athersclerotic plaque intoan unstable lesion; ascending aortic aneurysm-associated hypertension,hypercholesterolemia or diabetes mellitus; bicuspid aortic valve;Takayasu disease; rheumatoid arteritis; Marfan's syndrome; ankylosingspondylitis; giant cell arteritis; inflammatory aortic aneurysm;pulmonary artery aneurysm in Marfan's syndrome; aortic dissection in anaortic or peripheral large artery; angiogenesis; cancer; local discretelesion therapy; and arteriovenous malformation.

In some embodiments, the need is that the subject suffers from acardiovascular disorder, wherein the therapeutic effect isdown-regulation of an expression or activity of leptin in acardiovascular tissue.

In some embodiments, the cardiovascular tissue is aortic and/or mitralheart valve leaflet tissue. In some embodiments, the localadministration is effected by positioning a carrier capable of releasingthe leptin antagonist on an outer wall (e.g., tunica externa) or theinner wall (e.g., tunica intima) of an aorta.

In some embodiments, the cardiovascular tissue is arterial or venouswall tissue. In some embodiments, the local administration is effectedby positioning a carrier capable of releasing the leptin antagonist onan outer wall (e.g., tunica externa) or the inner wall (e.g., tunicaintima) of the arterial or venous wall tissue.

In some embodiments, the cardiovascular disorder is a vascular aneurysm.In some embodiments, the cardiovascular disorder is an aortic vasculardisorder. In some embodiments, the cardiovascular disorder is leftventricular remodeling.

In some embodiments, the local administration is effected via anintravascular catheter. In some embodiments, the local administration iseffected via direct injection.

According to an aspect of some embodiments of thee invention, there isalso provided a method for treatment of athersclerotic plaque,comprising: administering a pharmaceutically-effective amount of aleptin antagonist to athersclerotic plaque accumulated in the innerwalls of an artery, thereby at least one of: (a) reducing the rate and(b) reducing the incidence, of conversion of a stable atherscleroticplaque to an unstable lesion.

According to an aspect of some embodiments of the invention, there isalso provided a composition comprising a leptin antagonist and acarrier, for use in treating a disorder selected from the groupconsisting of: cardiovascular disease; remodeling of stableathersclerotic plaque into an unstable lesion; ascending aorticaneurysm-associated hypertension, hypercholesterolemia or diabetesmellitus; bicuspid aortic valve; Takayasu disease; rheumatoid arteritis;Marfan's syndrome; ankylosing spondylitis; giant cell arteritis;inflammatory aortic aneurysm; pulmonary artery aneurysm in Marfan'ssyndrome; aortic dissection in an aortic or peripheral large artery;site of arterial anastomosis, angiogenesis; cancer; local neoplasticdiscrete lesion therapy; and arteriovenous malformation, wherein thecarrier is configured for localized administration of the leptinantagonist.

In some embodiments, the carrier is a biodegradable support. In someembodiments, the biodegradable support is composed of a polymer selectedfrom the group consisting of a hydrogel, poly glycolic acid (PGA), polylactic co-glycolic acid (PLGA), polylactide (PLA), and poly (L-lactide)(PLLA), and combination thereof.

In some embodiments, the carrier is a hydrogel. In some embodiments, thecarrier is configured as a film. In some embodiments, the carrier is adevice selected from the group consisting of a mesh, a balloon and avascular graft. In some embodiments, the carrier is a depot-forminginjectable composition.

In some embodiments, the disorder is a cardiovascular disorder, whereinthe leptin antagonist effects down-regulation of an expression oractivity of leptin in a cardiovscular tissue. In some embodiments, thecardiovascular disorder is a vascular disorder. In some suchembodiments, the vascular disorder is an aortic vascular disorder. Insome embodiments, the cardiovascular disorder is left ventricularremodeling.

In some such embodiments, the cardiovascular tissue is aortic and/ormitral heart valve leaflet tissue. In some such embodiments, thecardiovascular tissue is arterial or venous wall tissue.

In some embodiments, the local administration is effected by positioninga carrier capable of releasing the leptin antagonist on a locationselected from the group consisting of: an outer wall of an aorta, anouter wall of an artery, an outer wall of an vein, a luminal surface ofan aorta, a luminal surface of an artery and a luminal surface of anvein. In some such embodiments, the local administration is effected bypositioning a carrier capable of releasing the leptin antagonist on anouter wall (e.g., tunica externa) or the inner wall (e.g., tunicaintima) of the arterial or venous wall tissue. In some embodiments, thelocalized administration is to be effected via an intravascularcatheter. In some embodiments, the localized administration is to beeffected via direct injection.

According to an aspect of some embodiments of the invention, there isalso provided a method of treating a condition in a subject in needthereof, the method comprising administering intracavitarily to innerwalls of a fluid-filled bodily cavity of the subject a compositioncomprising a leptin antagonist.

According to an aspect of some embodiments of the invention, there isalso provided a composition comprising: a leptin antagonist for use intreating a condition, wherein the composition is configured forintracavitary administration to inner walls of a fluid-filled bodilycavity of a subject.

According to an aspect of some embodiments of the invention, there isalso provided an intracavitarily-implantable medical device, comprising:at least one solid functional device part configured for deploying thedevice in a fluid-filled bodily cavity of a subject; and functionallyassociated with at least one the device component, a leptin antagonist.

According to an aspect of some embodiments of the invention, there isalso provided a surgical connecting device, comprising: a solid devicebody made of a material; and functionally associated with the devicebody, a pharmaceutically-effective amount of leptin antagonist. In someembodiments, the device body is in the form selected from the groupconsisting of surgical suture thread and a surgical staple.

Any suitable leptin antagonist may be used for implementing theteachings herein. Various types and specific suitable leptin antagonistsare listed in the description herein. In some embodiments, theantagonist is capable of binding a leptin receptor. In some embodiments,the leptin antagonist is incapable of dimerization. In some embodiments,the leptin antagonist comprises a poly peptide portion. In someembodiments, the leptin antagonist is selected from the group consistingof a polypeptide, a salt, and/or an ester thereof. In some embodiments,the leptin antagonist is a modified leptin polypeptide.

BRIEF DESCRIPTION OF THE FIGURES

Some embodiments of the invention are described herein with reference tothe accompanying figures. The description, together with the figures,makes apparent to a person having ordinary skill in the art how someembodiments of the invention may be practiced. The figures are for thepurpose of illustrative discussion and no attempt is made to showstructural details of an embodiment in more detail than is necessary fora fundamental understanding of the invention. For the sake of clarity,some objects depicted in the figures are not to scale.

In the Figures:

FIG. 1a-c illustrate a gel (FIG. 1a ), film (FIG. 1b ) and mesh (FIG. 1c) for local release of a leptin antagonist.

FIG. 2 illustrates a balloon catheter configured for local release of aleptin antagonist (drug release indicated by arrows).

FIG. 3 illustrates a slow release leptin eluted from a scaffold.

FIG. 4 illustrates the location of leptin film application on theanterior outer surface of the ascending aorta. Human arch angiogramdepicts mouse anatomy.

FIG. 5 illustrates a time course analysis of serum leptin level inApoE^(−/−) mice that underwent peri-aortic application of leptin film(20 μg).

FIG. 6 illustrates increased ascending aortic diameter at the locationof leptin film application versus controls.

FIG. 7 illustrates elastica staining and αSMA IHC analysis of ascendingaortic cross sections of mice locally treated with leptin versuscontrols.

FIG. 8 illustrates change in left ventricle (LV) wall thickness inleptin-treated (filled columns) versus control (open columns) mice.

FIG. 9 illustrates LV diameter in systole and diastole in leptin-treated(filled columns) and control (open columns) mice.

FIG. 10 illustrates LV fractional area change in leptin-treated (filledcolumns) versus control (open columns) mice.

FIG. 11 illustrates aortic and mitral valve leaflet thickness inleptin-treated and control mice.

FIG. 12 illustrates mean systolic blood pressure in angiotensin IItreated mice.

FIG. 13 illustrates a time course analysis presenting weight ofangiotensin II treated mice (open triangles), and mice receiving both,angiotensin II and leptin antagonist (LA).

FIG. 14 illustrates number of mice that succumbed due to rupturedabdominal and thoracic aneurysms in angiotensin II treated mice versusmice receiving angiotensin II and leptin antagonist (LA).

FIG. 15 illustrates ascending aortic dilatation in angiotensin IItreated mice versus mice receiving angiotensin II and leptin antagonist(LA).

FIG. 16 illustrates elastic lamella fragmentation and αSMA depletion inangiotensin II treated mice versus mice receiving angiotensin II andleptin antagonist (LA).

FIG. 17 illustrates leptin expression in medial SMCs (arrows) andmacrophages of atherosclerotic lesions (filled arrowheads) inangiotensin II treated, angiotensin II and leptin antagonist (LA)treated, and control mice.

FIG. 18 illustrates LV hypertrophy in angiotensin II treated mice versusmice receiving angiotensin II and leptin antagonist (LA).

FIG. 19 illustrates changes in LV diameter in angiotensin II treatedmice versus mice receiving angiotensin II and leptin antagonist (LA).

FIG. 20 illustrates LV fractional area change in angiotensin II treatedmice (open column) versus mice receiving angiotensin II and leptinantagonist (LA) (filled column).

FIG. 21 illustrates peak systolic velocity at the aortic valve inangiotensin II treated mice versus mice receiving angiotensin II andleptin antagonist (LA).

FIG. 22 illustrates aortic and mitral valve thickness (graph on left),and staining of valve leaflets with H&E (panels F-I). αSMA and TGFβ(panels J-M′, staining for aortic valves) in mice receiving angiotensinII versus mice treated with angiotensin II and leptin antagonist (LA).

FIG. 23 illustrates expression of leptin (D), and leptin receptor (E) innormal human aortic valve leaflet tissue.

FIG. 24 illustrates leptin and leptin receptor antigen prevalent insevere aortic valve stenosis, evident in SMC-like cells, andinfiltrating macrophages.

FIG. 25 illustrates leptin and leptin receptor mRNA levels in leafletsof stenosed aortic valve versus normal aortic valve controls, and fattissue (as positive control);

FIG. 26 illustrates proliferation of valve interstitial cells (VICs) inresponse to leptin stimulation; and

FIG. 27 schematically depicts embodiments of surgical connecting devicesaccording to the teachings herein;

FIG. 28 schematically depicts embodiments of the teachings hereinsuitable for intracavitary administration of leptin antagonist; and

FIG. 29 schematically depicts further embodiments of the teachingsherein suitable for intracavitary administration of leptin antagonist;and

DESCRIPTION OF SOME EMBODIMENTS

The invention, in some embodiments, relates to the field of medicine,and more particularly to methods and devices that use leptinantagonists. In some embodiments, the invention relates to compositionscomprising a leptin antagonist formulated for localized release of aleptin antagonist at the site of treatment as well as methods of usingsuch compositions for treating disorders, including cardiovasculardisorders. In some embodiments, the compositions comprising a leptinantagonist can be used for localized suppression of leptin-relatedconditions, including tissue remodeling processes.

Although it has been proposed that leptin might play a role in vascularinflammation, oxidative stress, and vascular smooth muscle hypertrophythat may contribute to coronary heart disease among other pathologies,to date no one has conclusively shown that localized down-regulation ofleptin activity can be used to treat cardiovascular disorderscharacterized by remodeling of cardiovascular tissue such as cardiac,arterial or valve tissue.

The present inventor set out to elucidate the role of leptin indisorders, such as cardiovascular disorders by employing a leptinantagonist in a localized manner. Experiments conducted by the presentinventor (see Examples section hereinbelow) demonstrate that localizedrelease of leptin in cardiovascular tissue can lead to cardiovasculartissue remodeling while localized down-regulation of leptin activity canlead to suppression and even reversal of cardiovascular tissue (arterialwall tissue, heart muscle tissue and valve leaflet tissue) remodelinginduced by angiotensin II. Thus, the present inventor has shown for thefirst time that a locally administered leptin antagonist can be used totreat cardiovascular disorders characterized by tissue remodeling.

While reducing the present invention to practice, the present inventorhas shown that down-regulation of leptin activity at specific sites inthe cardiovascular system can lead to suppression and reversal ofpathological tissue remodeling and thereby establishing localized leptindown-regulation as a suitable approach for treating variouscardiovascular disorders, such as cardiovascular disorders characterizedby pathological tissue remodeling.

The present inventor has discovered an unexpected pharmaceuticalefficacy of locally administered leptin antagonists, especially leptinantagonists administered by sustained release.

Particularly, the present inventor has found that in vivo implantationof a composition configured for sustained-release of leptin antagonistcan have a desirable pharmaceutical effect on tissue in proximity of theimplanted composition with limited or no substantial side-effects, forexample, no discernible hormonal or immunological effects.

The present inventor has also found that such in vivo administration(e.g., by implantation) inside a fluid-filled cavity of the body (forexample of the cardiovascular system such as blood vessels or cardiacchambers) can have a desirable pharmaceutical effect on tissue inproximity of the implanted composition with limited or no substantialside-effects: leptin antagonist from the composition has not been foundto be washed away by the fluid and, instead, has been found to interactwith the tissue providing a desirable pharmaceutical effect. Presumably,leptin antagonist released from the composition passes into and throughthe cavity walls (e.g., tunica intima) in pharmaceutically-effectiveamounts. This is particularly surprising in cavities of thecardiovascular system (e.g., veins, arteries and cardiac chambers) wherethe large volumes of blood passing through such cavities are expected towash away released leptin antagonist and where it is expected that thecardiovascular intima is relatively non-permeable to passage ofcompounds, especially proteins.

Without wishing to be held to any one theory, it is currently believedthat the success of some embodiments of the teachings herein is at leastpartially attributable to the serendipitous increased permeability ofcardiovascular intima during inflammation. It may be that the intimal ofhealthy cardiovascular intima is relatively impermeable to leptinantagonist released from the composition, so that there is little or nopassage of leptin antagonist into and through the endothelium andunderlying tissue, thereby avoiding substantial negative side-effects.In contrast, it seems that the permeability of the cardiovascular intimaduring inflammation increases sufficiently to allow passage of atherapeutically-effective amount of leptin antagonist released from thecomposition into the tissue. It is currently believed that this effectis self-regulating. A higher degree of inflammation leads to a higherdegree of intima permeability allowing passage of more leptin antagonistleading to a relatively high dose of leptin antagonist in the morepathological tissue. As inflammation decreases (inter alia, due to thepharmaceutical effect of the administered leptin antagonist), intimapermeability decreases thereby decreasing the dose of leptin antagonistactually in the tissue that is still sufficient to exercise a desiredpharmaceutical effect but with a reduced incidence of substantialnegative side effects.

In some embodiments, the present invention includes local administrationof leptin antagonist to treat and attenuate expansion of ascendingaortic aneurysm, and corresponding cardiac sequelae (driven by theaorto-ventricular coupling), including left ventricular hypertrophy, aswell as hyperplasia of left heart valve leaflets.

In some embodiments, the present invention includes treatment ofperipheral vascular disorders such as the progression of arterial orvenous aneurysms while minimizing systemic exposure to the administeredleptin antagonist.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

Thus, according to one aspect of the present invention there is provideda composition comprising a leptin antagonist and a carrier configuredfor localized administration, for treating disorders such ascardiovascular disorders. As used herein, “cardiovascular disorders”refer to disorders of the cardiovascular system, i.e. the heart andcentral, cranial and peripheral vasculature. Examples of such disordersinclude, but are not limited to valve stenosis, aneurysms, vesselresponse to vascular injury, cardiomyopathy and the like.

The carrier can be a solid, gel or liquid carrier, while the leptinantagonist can be any agent capable of down-regulating leptin activityin the target tissue. Examples of a leptin antagonist include agentscapable of binding and/or degrading leptin or leptin receptors as wellas agents capable of down-regulating leptin expression (at the DNA orRNA levels, i.e., agents capable of blocking transcription ortranslation). Specific preferred leptin antagonists are listedhereinbelow.

One example of a leptin antagonist that is an agent capable ofdown-regulating leptin is an antibody or antibody fragment capable ofspecifically binding leptin or a leptin receptor. Preferably, theantibody specifically binds at least one epitope of a leptin, e.g., anepitope defined amino acids 26-59 of mammalian leptin (e.g. ratleptin—SEQ ID NO 48). As used herein, the term “epitope” refers to anyantigenic determinant on an antigen to which the paratope of an antibodybinds.

As used herein, the term “antibody” refers to a substantially intactantibody molecule.

As used herein, the phrase “antibody fragment” refers to a functionalfragment of an antibody that is capable of binding to an antigen.

Suitable antibody fragments for practicing the present inventioninclude, inter alia, a complementarity-determining region (CDR) of animmunoglobulin light chain (referred to herein as “light chain”), a CDRof an immunoglobulin heavy chain (referred to herein as “heavy chain”),a variable region of a light chain, a variable region of a heavy chain,a light chain, a heavy chain, an Fd fragment, and antibody fragmentscomprising essentially whole variable regions of both light and heavychains such as an Fv, a single-chain Fv, an Fab, an Fab′, and anF(ab′)2.

Functional antibody fragments comprising whole or essentially wholevariable regions of both light and heavy chains are defined as follows:

(i) Fv, defined as a genetically engineered fragment consisting of thevariable region of the light chain and the variable region of the heavychain expressed as two chains;

(ii) single-chain Fv (“scFv”), a genetically engineered single-chainmolecule including the variable region of the light chain and thevariable region of the heavy chain, linked by a suitable polypeptidelinker.

(iii) Fab, a fragment of an antibody molecule containing a monovalentantigen-binding portion of an antibody molecule, obtained by treatingwhole antibody with the enzyme papain to yield the intact light chainand the Fd fragment of the heavy chain, which

(iv) Fab′, a fragment of an antibody molecule containing a monovalentantigen-binding portion of an antibody molecule, obtained by treatingwhole antibody with the enzyme pepsin, followed by reduction (two Fab′fragments are obtained per antibody molecule); and

(v) F(ab′)2, a fragment of an antibody molecule containing a monovalentantigen-binding portion of an antibody molecule, obtained by treatingwhole antibody with the enzyme pepsin (i.e., a dimer of Fab′ fragmentsheld together by two disulfide bonds).

Methods of generating monoclonal and polyclonal antibodies are wellknown in the art. Antibodies may be generated via any one of severalknown methods, which may employ induction of in vivo production ofantibody molecules, screening of immunoglobulin libraries (Orlandi, R.et al. (1989). Cloning immunoglobulin variable domains for expression bythe polymerase chain reaction. Proc Natl Acad Sci USA 86, 3833-3837; andWinter, G. and Milstein, C. (1991). Man-made antibodies. Nature 349,293-299), or generation of monoclonal antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique, the human B-cell hybridoma technique, and theEpstein-Barr virus (EBV)-hybridoma technique (Kohler, G. and Milstein,C. (1975). Continuous cultures of fused cells secreting antibody ofpredefined specificity. Nature 256, 495-497; Kozbor, D. et al. (1985).Specific immunoglobulin production and enhanced tumorigencity followingascites growth of human hybridomas. J Immunol Methods 81, 31-42; Cote RJ. et al. (1983). Generation of human monoclonal antibodies reactivewith cellular antigens. Proc Natl Acad Sci USA 80, 2026-2030; and Cole,S. P. et al. (1984). Human monoclonal antibodies. Mol Cell Biol 62,109-120).

In cases where target antigens are too small to elicit an adequateimmunogenic response, such antigens (referred to as “haptens”) can becoupled to antigenically neutral carriers such as keyhole limpethemocyanin (KLH) or serum albumin (e.g., bovine serum albumin (BSA))carriers (see, for example, U.S. Pat. Nos. 5,189,178 and 5,239,078).Coupling a hapten to a carrier can be effected using methods well knownin the art. For example, direct coupling to amino groups can be effectedand optionally followed by reduction of the imino linkage formed.Alternatively, the carrier can be coupled using condensing agents suchas dicyclohexyl carbodiimide or other carbodiimide dehydrating agents.Linker compounds can also be used to effect the coupling; bothhomobifunctional and heterobifunctional linkers are available fromPierce Chemical Company, Rockford, Ill., USA. The resulting immunogeniccomplex can then be injected into suitable mammalian subjects such asmice, rabbits, and others. Suitable protocols involve repeated injectionof the immunogen in the presence of adjuvants according to a scheduledesigned to boost production of antibodies in the serum. The titers ofthe immune serum can readily be measured using immunoassay procedureswhich are well known in the art.

The antisera obtained can be used directly or monoclonal antibodies maybe obtained, as described hereinabove.

Antibody fragments may be obtained using methods well known in the art.(See, for example, Harlow, E. and Lane, D. (1988). Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, New York.) Forexample, antibody fragments according to the present invention can beprepared by proteolytic hydrolysis of the antibody or by expression inE. coli or mammalian cells (e.g., Chinese hamster ovary (CHO) cellculture or other protein expression systems) of DNA encoding thefragment.

Alternatively, antibody fragments can be obtained by pepsin or papaindigestion of whole antibodies by conventional methods. As describedhereinabove, an (Fab′)₂ antibody fragments can be produced by enzymaticcleavage of antibodies with pepsin to provide a 5S fragment. Thisfragment can be further cleaved using a thiol reducing agent, andoptionally a blocking group for the sulfhydryl groups resulting fromcleavage of disulfide linkages, to produce 3.5S Fab′ monovalentfragments. Alternatively, enzymatic cleavage using pepsin produces twomonovalent Fb′ fragments and an Fc fragment directly. Ample guidance forpracticing such methods is provided in the literature of the art (forexample, refer to: U.S. Pat. Nos. 4,036,945 and 4,331,647; and Porter,R. R. (1959). The hydrolysis of rabbit γ-globulin and antibodies withcrystalline papain. Biochem J 73, 119-126). Other methods of cleavingantibodies, such as separation of heavy chains to form monovalentlight-heavy chain fragments, further cleavage of fragments, or otherenzymatic, chemical, or genetic techniques may also be used, so long asthe fragments retain the ability to bind to the antigen that isrecognized by the intact antibody.

As described hereinabove, an Fv is composed of paired heavy chainvariable and light chain variable domains. This association may benoncovalent (see, for example, Inbar, D. et al. (1972). Localization ofantibody-combining sites within the variable portions of heavy and lightchains. Proc Natl Acad Sci USA 69, 2659-2662). Alternatively, asdescribed hereinabove, the variable domains may be linked to generate asingle-chain Fv by an intermolecular disulfide bond, or alternately suchchains may be cross-linked by chemicals such as glutaraldehyde.

Preferably, the Fv is a single-chain Fv. Single-chain Fvs are preparedby constructing a structural gene comprising DNA sequences encoding theheavy chain variable and light chain variable domains connected by anoligonucleotide encoding a peptide linker. The structural gene isinserted into an expression vector, which is subsequently introducedinto a host cell such as E. coli. The recombinant host cells synthesizea single polypeptide chain with a linker peptide bridging the twovariable domains. Ample guidance for producing single-chain Fvs isprovided in the literature of the art (see, e.g.: Whitlow, M. andFilpula, D. (1991). Single-chain Fv proteins and their fusion proteins.METHODS: A Companion to Methods in Enzymology 2(2), 97-105; Bird, R. E.et al. (1988). Single-chain antigen-binding proteins. Science 242,423-426; Pack, P. et al. (1993). Improved bivalent miniantibodies, withidentical avidity as whole antibodies, produced by high cell densityfermentation of Escherichia coli, Biotechnology (N.Y.) 11(11),1271-1277; and U.S. Pat. No. 4,946,778).

Isolated complementarity-determining region peptides can be obtained byconstructing genes encoding the CDR of an antibody of interest. Suchgenes may be prepared, for example, by RT-PCR of the mRNA of anantibody-producing cell. Ample guidance for practicing such methods isprovided in the literature of the art (e.g., Larrick, J. W. and Fry, K.E. (1991). PCR Amplification of Antibody Genes. METHODS: A Companion toMethods in Enzymology 2(2), 106-110).

It will be appreciated that for human therapy, humanized antibodies arepreferred. Humanized forms of non-human (e.g., murine) antibodies aregenetically engineered chimeric antibodies or antibody fragments having(preferably minimal) portions derived from non-human antibodies.Humanized antibodies include antibodies in which the CDRs of a humanantibody (recipient antibody) are replaced by residues from a CDR of anon-human species (donor antibody), such as mouse, rat, or rabbit,having the desired functionality. In some instances, the Fv frameworkresidues of the human antibody are replaced by corresponding non-humanresidues. Humanized antibodies may also comprise residues found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDRs correspond to those of anon-human antibody and all or substantially all of the framework regionscorrespond to those of a relevant human consensus sequence. Humanizedantibodies optimally also include at least a portion of an antibodyconstant region, such as an Fc region, typically derived from a humanantibody (see, for example: Jones, P. T. et al. (1986). Replacing thecomplementarity-determining regions in a human antibody with those froma mouse. Nature 321, 522-525; Riechmann, L. et al. (1988). Reshapinghuman antibodies for therapy. Nature 332, 323-327; Presta, L. G.(1992b). Curr Opin Struct Biol 2, 593-596; and Presta, L. G. (1992a).Antibody engineering. Curr Opin Biotechnol 3(4), 394-398).

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as imported residues, whichare typically taken from an imported variable domain. Humanization canbe performed essentially as described (see, for example: Jones et al.(1986); Riechmann et al. (1988); Verhoeyen, M. et al. (1988). Reshapinghuman antibodies: grafting an antilysozyme activity. Science 239,1534-1536; and U.S. Pat. No. 4,816,567), by substituting human CDRs withcorresponding rodent CDRs. Accordingly, humanized antibodies arechimeric antibodies, wherein substantially less than an intact humanvariable domain has been substituted by the corresponding sequence froma non-human species. In practice, humanized antibodies may be typicallyhuman antibodies in which some CDR residues and possibly some frameworkresidues are substituted by residues from analogous sites in rodentantibodies.

Human antibodies can also be produced using various additionaltechniques known in the art, including phage-display libraries(Hoogenboom, H. R. and Winter, G. (1991). By-passing immunization. Humanantibodies from synthetic repertoires of germline VH gene segmentsrearranged in vitro. J Mol Biol 227, 381-388; Marks, J. D. et al.(1991). By-passing immunization. Human antibodies from V-gene librariesdisplayed on phage. J Mol Biol 222, 581-597; Cole et al. (1985),Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96;and Boerner, P. et al. (1991). Production of antigen-specific humanmonoclonal antibodies from in vitro-primed human splenocytes. J Immunol147, 86-95). Humanized antibodies can also be created by introducingsequences encoding human immunoglobulin loci into transgenic animals,e.g., into mice in which the endogenous immunoglobulin genes have beenpartially or completely inactivated. Upon antigenic challenge, humanantibody production is observed in such animals which closely resemblesthat seen in humans in all respects, including gene rearrangement, chainassembly, and antibody repertoire. Ample guidance for practicing such anapproach is provided in the literature of the art (for example, refer toU.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425;and 5,661,016; Marks, J.D. et al. (1992). By-passing immunization:building high affinity human antibodies by chain shuffling.Biotechnology (N.Y.) 10(7), 779-783; Lonberg et al., 1994. Nature368:856-859; Morrison, S. L. (1994). News and View: Success inSpecification. Nature 368, 812-813; Fishwild, D. M. et al. (1996).High-avidity human IgG kappa monoclonal antibodies from a novel strainof minilocus transgenic mice. Nat Biotechnol 14, 845-851; Neuberger, M.(1996). Generating high-avidity human Mabs in mice. Nat Biotechnol 14,826; and Lonberg, N. and Huszar, D. (1995). Human antibodies fromtransgenic mice. Int Rev Immunol 13, 65-93.)

After antibodies have been obtained, they may be tested for activity,for example via enzyme-linked immunosorbent assay (ELISA).

Anti-leptin antibodies as well as epitope sequences suitable forgenerating antibodies and antibody fragments are described inUS20070104708 (SEQ ID NOs 49-55) which is incorporated herein byreference as if fully set-forth herein.

Leptin peptide antagonists can also be used with the present invention.One leptin antagonist, a modified mammalian leptin polypeptide termedsuperactive leptin mutein is disclosed in U.S. Pat. No. 8,969,292 whichis incorporated by reference as if fully set-forth herein.

The term “peptide” as used herein encompass native peptides (eitherdegradation products, synthetically synthesized peptides, or recombinantpeptides), peptidomimetics (typically, synthetically synthesizedpeptides), and the peptide analogues peptoids and semipeptoids, and mayhave, for example, modifications rendering the peptides more stablewhile in a body or more capable of penetrating into cells. Suchmodifications include, but are not limited to: N-terminus modifications;C-terminus modifications; peptide bond modifications, including but notlimited to CH₂—NH, CH₂—S, CH₂—S═O, O═C—NH, CH₂—O, CH₂—CH₂, S═C—NH,CH═CH, and CF═CH; backbone modifications; and residue modifications.Methods for preparing peptidomimetic compounds are well known in the artand are specified, for example, in Ramsden, C. A., ed. (1992),Quantitative Drug Design, Chapter 17.2, F. Choplin Pergamon Press, whichis incorporated by reference as if fully set forth herein. Furtherdetails in this respect are provided hereinbelow.

Peptide bonds (—CO—NH—) within the peptide may be substituted, forexample, by N-methylated bonds (—N(CH3)—CO—); ester bonds(—C(R)H—C—O—O—C(R)—N—); ketomethylene bonds (—CO—CH2-); a_-aza bonds(—NH—N(R)—CO—), wherein R is any alkyl group, e.g., methyl; carba bonds(—CH2-NH—); hydroxyethylene bonds (—CH(OH)—CH2-); thioamide bonds(—CS—NH—); olefinic double bonds (—CH═CH—); retro amide bonds (—NH—CO—);and peptide derivatives (—N(R)—CH2-CO—), wherein R is the “normal”sidechain, naturally presented on the carbon atom. These modifications canoccur at any of the bonds along the peptide chain and even at several(2-3) at the same time.

Natural aromatic amino acids, Trp, Tyr, and Phe, may be substituted forsynthetic non-natural acids such as, for instance,tetrahydroisoquinoline-3-carboxylic acid (TIC), naphthylelanine (NoI),ring-methylated derivatives of Phe, halogenated derivatives of Phe, ando-methyl-Tyr.

In addition to the above, the peptides of the present invention may alsoinclude one or more modified amino acids or one or more non-amino acidmonomers (e.g., fatty acids, complex carbohydrates, etc.)

The term “amino acid” or “amino acids” is understood to include the 20naturally occurring amino acids; those amino acids often modifiedpost-translationally in vivo, including, for example, hydroxyproline,phosphoserine, and phosphothreonine; and other less common amino acids,including but not limited to 2-aminoadipic acid, hydroxylysine,isodesmosine, nor-valine, nor-leucine, and ornithine. Furthermore, theterm “amino acid” includes both D- and L-amino acids.

Amino acids are referred to by the standard three letter code. Aminoacids are L amino acids unless otherwise noted, for example, by additionof the prefix “D”. For example, the code Trp refers to L-tryptophan,while the codes D-Trp and DTrp refers to D-tryptophan. The code Aibrefers to 2-aminoisobutyric acid. The code Orn refers to ornithine. Thecode Lys-Ac refers to acetyllysine. The code HomoLys refers tohomolysine. The code H-Cys refers to homocysteine.

In some embodiments, peptidic leptin antagonists used to implement theteachings herein are utilized in a linear form, although in someembodiments, cyclic forms thereof are used.

In some embodiments, peptidic leptin antagonists used to implement theteachings herein are synthesized by any techniques that are known tothose skilled in the art of peptide synthesis. For solid phase peptidesynthesis, a summary of the many techniques may be found in: Stewart, J.M. and Young, J. D. (1963), “Solid Phase Peptide Synthesis,” W. H.Freeman Co. (San Francisco); and Meienhofer, J (1973). “HormonalProteins and Peptides, ” vol. 2, p. 46, Academic Press (New York). For areview of classical solution synthesis, see Schroder, G. and Lupke, K.(1965). The Peptides, vol. 1, Academic Press (New York).

In general, peptide synthesis methods comprise the sequential additionof one or more amino acids or suitably protected amino acids to agrowing peptide chain. Normally, either the amino or the carboxyl groupof the first amino acid is protected by a suitable protecting group. Theprotected or derivatized amino acid can then either be attached to aninert solid support or utilized in solution by adding the next aminoacid in the sequence having the complimentary (amino or carboxyl) groupsuitably protected, under conditions suitable for forming the amidelinkage. The protecting group is then removed from this newly addedamino acid residue and the next amino acid (suitably protected) is thenadded, and so forth; traditionally this process is accompanied by washsteps as well. After all of the desired amino acids have been linked inthe proper sequence, any remaining protecting groups (and any solidsupport) are removed sequentially or concurrently, to afford the finalpeptide compound. By simple modification of this general procedure, itis possible to add more than one amino acid at a time to a growingchain, for example, by coupling (under conditions which do not racemizechiral centers) a protected tripeptide with a properly protecteddipeptide to form, after deprotection, a pentapeptide, and so forth.

Further description of peptide synthesis is disclosed in U.S. Pat. No.6,472,505. A preferred method of preparing the peptide compounds of thepresent invention involve solid-phase peptide synthesis, utilizing asolid support.

In some embodiments, peptidic leptin antagonists used to implement theteachings herein are generated using cell expression approaches byutilizing expression vectors for prokaryotic or eukaryotic expression oralternatively, the peptide can be expressed in-situ by delivering asuitable expression construct to cardiovascular tissue.

To express the peptide sequence in cardiovascular cells, apolynucleotide sequence encoding the peptide (see, for example,US20130133089) is preferably ligated into a nucleic acid constructsuitable for mammalian cell expression. Such a nucleic acid constructincludes a promoter sequence for directing transcription of thepolynucleotide sequence in the cell in a constitutive or induciblemanner.

Constitutive promoters suitable for use with the present invention arepromoter sequences that are active under most environmental conditionsand most types of cells, such as the cytomegalovirus (CMV) and Roussarcoma virus (RSV).

Polyadenylation sequences can also be added to the expression vector inorder to increase the efficiency of mRNA translation. Two distinctsequence elements are required for accurate and efficientpolyadenylation: GU- or U-rich sequences located downstream from thepolyadenylation site and a highly conserved sequence of six nucleotides,namely AAUAAA, located 11-30 nucleotides upstream of the site.Termination and polyadenylation signals suitable for the presentinvention include those derived from SV40.

In addition to the embodiments already described, the expression vectorof the present invention may typically contain other specializedelements intended to increase the level of expression of cloned nucleicacids or to facilitate the identification of cells that carry therecombinant DNA. For example, a number of animal viruses contain DNAsequences that promote extra-chromosomal replication of the viral genomein permissive cell types. Plasmids bearing these viral replicons arereplicated episomally as long as the appropriate factors are provided bygenes either carried on the plasmid or with the genome of the host cell.

The expression vector of the present invention may or may not include aeukaryotic replicon. If a eukaryotic replicon is present, the vector iscapable of amplification in eukaryotic cells using the appropriateselectable marker. If the vector does not comprise a eukaryoticreplicon, no episomal amplification is possible.

Examples for mammalian expression vectors include, but are not limitedto, pcDNA3 (SEQ ID NO 56), pc DNA3.1 (+/−) (SEQ ID NO 57-58), pGL3 (SEQID NO 59), pZeoSV2 (+/−) (SEQ ID NO 60), pSecTag2 (SEQ ID NO 61),pDisplay (SEQ ID NO 62), pEF/myc/cyto (SEQ ID NO 63), pCMV/myc/cyto (SEQID NO 64), pCR3.1 (SEQ ID NO 65), pSinRep5 (SEQ ID NO 66), DH26S, DHBB,pNMT1, pNMT41, and pNMT81, which are available from Invitrogen, pCI (SEQID NO 67) which is available from Promega, pMbac (SEQ ID NO 68), pPbac(SEQ ID NO 69), pBK-RSV (SEQ ID NO 70) and pBK-CMV (SEQ ID NO 71), whichare available from Strategene, pTRES which is available from Clontech,and their derivatives.

Expression vectors containing regulatory elements from eukaryoticviruses such as retroviruses can be also used. SV40 vectors includepSVT7 and pMT2 (SEQ ID NO 72), for instance. Vectors derived from bovinepapilloma virus include pBV-lMTHA, and vectors derived from Epstein-Barrvirus include pHEBO and p2O5. Other exemplary vectors include pMSG,pAV009/A⁺, pMTO10/A⁺, pMAMneo-5, baculovirus pDSVE, and any other vectorallowing expression of proteins under the direction of the SV40 earlypromoter, SV40 later promoter, metallothionein promoter, murine mammarytumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter,or other promoters shown effective for expression in eukaryotic cells.

Viruses are very specialized infectious agents that have evolved, inmany cases, to elude host defense mechanisms. Typically, viruses infectand propagate in specific cell types. The targeting specificity of viralvectors utilizes its natural specificity to specifically targetpredetermined cell types and thereby introduce a recombinant gene intothe infected cell. Thus, the type of vector used by the presentinvention will depend on the cell type transformed. The ability toselect suitable vectors according to the cell type transformed is wellwithin the capabilities of the ordinarily skilled artisan and as such,no general description of selection considerations is provided herein.For example, bone marrow cells can be targeted using the human T-cellleukemia virus type I (HTLV-1) and kidney cells may be targeted usingthe heterologous promoter present in the baculovirus Autographacalifornica multiple nucleopolyhedrovirus (AcMNPV), as described byLiang, C. Y. et al. (2004). High efficiency gene transfer into mammaliankidney cells using baculovirus vectors. Arch Virol 149, 51-60.

Recombinant viral vectors are useful for in vivo expression of a leptinpeptide since they offer advantages such as lateral infection andtargeting specificity. Lateral infection is inherent in the life cycleof retrovirus, for example, and is the process by which a singleinfected cell produces many progeny virions that bud off and infectneighboring cells. The result is the rapid infection of a large area ofcells, most of which were not initially infected by the original viralparticles. This is in contrast to vertical-type infection in which theinfectious agent spreads only through daughter progeny. Viral vectorscan also be produced that are unable to spread laterally. Thischaracteristic can be useful if the desired purpose is to introduce aspecified gene into only a localized number of targeted cells.

As is mentioned hereinabove, compositions (also called,compositions-of-matter) according to the teachings herein also includesa carrier for local delivery of the leptin antagonist. Such a carriercan be a mesh (FIG. 1c ) an injectable gel (e.g. in-situ forming depot)(FIG. 1a ), a thin (preferably biodegradable) film (FIG. 1b ), ascaffold (FIG. 3). In some embodiments, the composition is a coating ona medical device (FIG. 27A). In some embodiments, a medical device isimpregnated with the composition (FIG. 27B). In some embodiments, thecomposition is in the form of a sheet (such as the film of FIG. 1b orthe mesh of FIG. 1c ) that constitutes a portion of a medical devicesuch as a stent cover (FIG. 27C) or graft of a graft-stent assembly(FIG. 27D). In some embodiments, the composition constitutes a medicaldevice (FIG. 27E).

In some embodiments a carrier of a composition is a balloon catheter, ora composition is delivered locally using a drug-eluting balloon catheter(FIG. 2). The manufacture and use of drug-eluting balloons for localizeddelivery of active-pharmaceutical ingredients are well known in the art(especially to the walls of fluid-filled bodily cavities, such as of thecardiovascular system), for example, the In.Pact Admiral® DCBdrug-coated balloon by Medtronic (Dublin, Ireland) and Lutonix® 035 byC. R. Bard, Inc. (Murray Hill, N.J., USA).

Examples of in-situ formed depots (ISFD) include semi-solid polymerswhich can be injected as a melt and form a depot upon cooling to bodytemperature or tow part systems which gel upon mixing (FIG. 3a ).Depending on the embodiments, such compositions can be injected into orin contact with bodily tissue that is to be treated

The requirements for a semi-solid ISFDs include low melting or glasstransition temperatures in the range of 25-65° C. and an intrinsicviscosity in the range of 0.05-0.8 dl/g [12-14]. Below the viscositythreshold of 0.05 dl/g no delayed diffusion could be observed, whereasabove 0.8 dl/g the ISFD was no longer injectable using a needle. Atinjection temperatures above 37° C. but below 65° C. these polymersbehave like viscous fluids which solidify to highly viscous depots.Drugs are incorporated into the molten polymer by mixing without theapplication of solvents. In the art, it is known to use thermoplasticpastes (TP) can be used to generate a subcutaneous drug reservoir fromwhich diffusion occurs into the systemic circulation. In contrast, insome embodiments of the teachings herein, a thermoplastic paste is usedto generate a composition for the sustained release of leptin antagonistfrom which diffusion occurs into tissue in contact with the composition,thereby effecting sustained-release local administration of the leptinantagonist.

In situ cross-linked polymer systems utilize a cross-linked polymernetwork to control the diffusion of bioactive agents (e.g., leptinantagonists for implementing the teachings herein) over a prolongedperiod of time, thereby allowing implementation of sustained releasecompositions comprising leptin antagonists for use in localadministration thereof. Use of in situ cross-linking implantsnecessitate protection of the bioactive agents during the cross-linkingreaction. This could be achieved by encapsulation into fast degradinggelatin micro-particles.

An ISFD can also be based on polymer precipitation. A water-insolubleand biodegradable polymer is dissolved in a biocompatible organicsolvent to which leptin antagonist is added forming a solution orsuspension after mixing that constitutes a composition according to theteachings herein. When this composition is injected into the body of asubject in need thereof the water miscible organic solvent dissipatesand water penetrates into the organic phase. This leads to phaseseparation and precipitation of the polymer forming a depot at the siteof injection. One example of such a system is Atrigele™ (ARTIXLaboratories). The thus-formed depot is a composition for the sustainedrelease of leptin antagonist from which diffusion occurs into tissue incontact with the composition, thereby effecting sustained-release localadministration of the leptin antagonist.

Thermally induced gelling systems can also be used as ISFDs. Numerouspolymers show abrupt changes in solubility as a function ofenvironmental temperature. The prototypic thermosensitive polymer ispoly(N-isopropyl acryl amide), poly-NIPAAM, which exhibits a rathersharp lower critical solution temperature.

Thermoplastic pastes such as the new generation of poly(ortho-esters)developed by AP Pharma can also be used for depot drug delivery. Suchpastes include polymers that are semi-solid at room temperature, henceheating for drug incorporation and injection is no longer necessary.Injection is possible through needles no larger than 22 gauge. Theleptin antagonist is mixed into the systems in a dry and, therefore,stabilized state. Shrinkage or swelling upon injection is thought to bemarginal and, therefore, the initial drug burst is expected to be lowerthan in the other types of ISFD. An additional advantage is afforded bythe self-catalyzed degradation by surface erosion. As noted above, IFSDcompositions are suitable for effecting sustained-release localadministration of the leptin antagonist. In some embodiments, an IFSDcomposition can be formulated for sustained-release (SR),extended-release (ER, XR, or XL), time-release or timed-release,controlled-release (CR), or continuous-release.

Examples of thin films (FIG. 3b ) suitable for release of a leptinantagonist (or polynucleotide encoding same) include polymeric films(for a review of thin films, see Zelikin ACS Nano, 2010, 4 (5), pp2494-2509; Venkat et al. 2010, Polymer Thin Films for BiomedicalApplications, Wiley VCH Verlag GmbH & Co. KGaA, Weinheim). Such thinfilm carriers can be biodegradable or dissolvable over time.

Biodegradable microspheres fabricated from, for example, PGA, PLGA, PLA,or PLLA can also be used for local delivery of a leptin antagonist. Suchmicrospheres can be produced as described by Kim and Park (J ControlRelease, 2004 Jul. 23;98(1):115-25).

A balloon such as an angioplasty balloon (FIG. 2) can also be used todeliver a leptin antagonist to a vascular wall or an inner wall of aheart chamber. Approach for coating/loading a balloon with a peptide aredescribed in EP2643030; U.S. Pat. Nos. 8,617,136; 8,617,104; 8,617,114;WO1997017099; US20110166547 and US20120150142. As noted above, suchdrug-eluting balloons for use for localized delivery ofactive-pharmaceutical ingredients are well known in the art, forexample, the In.Pact Admiral® drug-coated balloon by Medtronic (Dublin,Ireland) and Lutonix® 035 by C. R. Bard, Inc. (Murray Hill, N.J., USA).It is important to note that such drug-eluting balloons are known toadminister extended release compositions of active pharmaceuticalingredients, e.g., In.Pact Admiral delivers a composition that providesextended release of a continuous therapeutic dose of Paclitaxel for over180 days.

Although delivery of leptin or leptin receptor binding agents such asthose described above (or expression thereof in cardiovascular cells),is presently preferred, downregulation of leptin activity at specifictissues can also be effected at the transcript level using a variety ofmolecules that interfere with transcription and/or translation (e.g.,antisense, siRNA, Ribozyme, or DNAzyme).

RNA interference can be used to downregulate endogenous leptin via asmall interfering RNA (siRNA) molecule. RNAi is a two-step process, inthe first, the initiation step, input double-stranded (dsRNA) isdigested into 21- to 23-nucleotide (nt) small interfering RNAs (siRNAs),probably by the action of Dicer, a member of the RNase III family ofdsRNA-specific ribonucleases, which processes (cleaves) dsRNA(introduced directly or by means of a transgene or a virus) in anATP-dependent manner. Successive cleavage events degrade the RNA to 19-to 21-bp duplexes (the siRNA), each with 2-nucleotide 3′ overhangs(Hutvagner, G. and Zamore, P. D. (2002), RNAi: Nature abhors adouble-strand. Curr Opin Gen Dev 12, 225-232; and Bernstein, E. (2001).Role for a bidentate ribonuclease in the initiation step of RNAinterference. Nature 409, 363-366).

In the second step, termed the effector step, the siRNA duplexes bind toa nuclease complex to form the RNA-induced silencing complex (RISC). AnATP-dependent unwinding of the siRNA duplex is required for activationof the RISC. The active RISC then targets the homologous transcript bybase-pairing interactions and cleaves the mRNA into 12-nucleotidefragments from the 3′ terminus of the siRNA (Hutvagner and Zamore(2002); Hammond et al. (2001) Nat. Rev. Gen. 2:110-119 (2001); andSharp, P. A. (2001). RNA interference. Genes Dev 15, 485-490). Althoughthe mechanism of cleavage remains to be elucidated, research indicatesthat each RISC contains a single siRNA and an RNase (Hutvagner andZamore (2002)).

Synthesis of RNAi molecules suitable for use with the present inventioncan be effected as follows. First, the leptin mRNA sequence (SEQ ID NO2) is scanned downstream of the AUG start codon for AA-dinucleotidesequences. Occurrence of each AA and the 19 3′-adjacent nucleotides isrecorded as a potential siRNA target site. Preferably, siRNA targetsites are selected from the open reading frame (ORF), as untranslatedregions (UTRs) are richer in regulatory protein binding sites.UTR-binding proteins and/or translation initiation complexes mayinterfere with binding of the siRNA endonuclease complex (Tuschl(2001)). It will be appreciated, however, that siRNAs directed atuntranslated regions may also be effective, as demonstrated for GAPDH,wherein siRNA directed at the 5′ UTR mediated about a 90% decrease incellular GAPDH mRNA and completely abolished protein levels(wwwdotambiondotcom/techlib/tn/91/912dothtml).

Second, potential target sites are compared to an appropriate genomicdatabase (e.g., human, mouse, rat, etc.) using any sequence alignmentsoftware, such as the BlastN software available from the NCBI server(wwwdotncbidotnlmdotnihdotgov/BLAST/). Putative target sites thatexhibit significant homology to other coding sequences are filtered out.

Qualifying target sequences are selected as templates for siRNAsynthesis. Preferred sequences are those including low G/C content, asthese have proven to be more effective in mediating gene silencing ascompared with sequences including G/C content higher than 55%. Severaltarget sites are preferably selected along the length of the target genefor evaluation. For better evaluation of the selected siRNAs, a negativecontrol is preferably used in conjunction. Negative-control siRNAspreferably include the same nucleotide composition as the siRNAs butlack significant homology to the genome. Thus, a scrambled nucleotidesequence of the siRNA is preferably used, provided it does not displayany significant homology to any other gene.

Another agent capable of downregulating leptin is a DNAzyme molecule,which is capable of specifically cleaving an mRNA transcript or a DNAsequence of the leptin. DNAzymes are single-stranded polynucleotidesthat are capable of cleaving both single- and double-stranded targetsequences (Breaker, R. R. and Joyce, G. F. (1995). A DNA enzyme withMg²⁺-dependent RNA phosphoesterase activity, Curr Biol 2, 655-660;Santoro, S. W. and Joyce, G. F. (1997). A general purpose RNA-cleavingDNA enzyme, Proc Natl Acad Sci USA 94, 4262-4266). A general model (the“10-23” model) for the DNAzyme has been proposed. “10-23” DNAzymes havea catalytic domain of 15 deoxyribonucleotides, flanked by twosubstrate-recognition domains of seven to nine deoxyribonucleotideseach. This type of DNAzyme can effectively cleave its substrate RNA atpurine; pyrimidine junctions (Santoro and Joyce (1997)); for review ofDNAzymes, see; Khachigian, L. M. (2002). DNAzymes: cutting a path to anew class of therapeutics. Curr Opin Mol Ther 4, 119-121.

Examples of construction and amplification of synthetic, engineeredDNAzymes recognizing single- and double-stranded target cleavage sitesare disclosed in U.S. Pat. No. 6,326,174 to Joyce et al. DNAzymes ofsimilar design directed against the human Urokinase receptor wererecently observed to inhibit Urokinase receptor expression, andsuccessfully inhibit colon cancer cell metastasis in vivo (Itoh, T. etal., Abstract 409, American Society of Gene Therapy 5th Annual Meeting(wwwdotasgtdotorg), Jun. 5-9, 2002, Boston, Mass. USA.). In anotherapplication, DNAzymes complementary to ber-abl oncogenes were successfulin inhibiting the oncogene's expression in leukemia cells, and inreducing relapse rates in autologous bone marrow transplants in cases ofChronic Myelogenous Leukemia (CML) and Acute Lymphoblastic Leukemia(ALL).

Downregulation of leptin can also be effected by using an antisensepolynucleotide capable of specifically hybridizing with an mRNAtranscript encoding leptin.

Design of antisense molecules that can be used to efficientlydownregulate a leptin must be effected while considering two aspectsimportant to the antisense approach. The first aspect is delivery of theoligonucleotide into the cytoplasm of the appropriate cells, while thesecond aspect is design of an oligonucleotide that specifically bindsthe designated mRNA within cells in a manner inhibiting the translationthereof.

The prior art teaches of a number of delivery strategies which can beused to efficiently deliver oligonucleotides into a wide variety of celltypes (see, for example: Luft, F. C. (1998). Making sense out ofantisense oligodeoxynucleotide delivery: getting there is half the fun.J Mol Med 76(2), 75-76 (1998); Kronenwett et al. (1998).Oligodeoxyribonucleotide uptake in primary human hematopoietic cells isenhanced by cationic lipids and depends on the hematopoietic cellsubset. Blood 91, 852-862; Rajur, S. B. et al. (1997). Covalentprotein-oligonucleotide conjugates for efficient delivery of antisensemolecules. Bioconjug Chem 8, 935-940; Lavigne et al. Biochem Biophys ResCommun 237: 566-71 (1997); and Aoki, M. et al. (1997). In vivo transferefficiency of antisense oligonucleotides into the myocardium usingHVJ-liposome method. Biochem Biophys Res Commun 231, 540-545).

In addition, also available are algorithms for identifying thosesequences with the highest predicted binding affinity for their targetmRNA based on a thermodynamic cycle that accounts for the energetics ofstructural alterations in both the target mRNA and the oligonucleotide(see, for example, Walton, S. P. et al. (199). Prediction of antisenseoligonucleotide binding affinity to a structured RNA target. BiotechnolBioeng 65, 1-9).

Such algorithms have been successfully used to implement an antisenseapproach in cells. For example, the algorithm developed by Walton et al.enabled scientists to successfully design antisense oligonucleotides forrabbit beta-globin (RBG) and mouse tumor necrosis factor-alpha(TNF-alpha) transcripts. The same research group has more recentlyreported that the antisense activity of rationally selectedoligonucleotides against three model target mRNAs (human lactatedehydrogenase A and B and rat gp130) in cell culture as evaluated by akinetic PCR technique proved effective in almost all cases, includingtests against three different targets in two cell types withphosphodiester and phosphorothioate oligonucleotide chemistries.

In addition, several approaches for designing and predictingefficiencies of specific oligonucleotides using an in vitro system werealso published (Matveeva, O. et al. (1998). Prediction of antisenseoligonucleotide efficacy by in vitro methods, Nature Biotechnology 16,1374-1375).

Another agent capable of down-regulating leptin is a ribozyme moleculecapable of specifically cleaving an mRNA transcript encoding leptin is aribozyme molecule capable of specifically cleaving an mRNA transcriptencoding leptin. Ribozymes increasingly are being used for thesequence-specific inhibition of gene expression by the cleavage of mRNAsencoding proteins of interest (Welch, P. J. et al. (1998). Expression ofribozymes in gene transfer systems to modulate target RNA levels. CurrOpin Biotechnol 9, 486-496).

An additional method of regulating the expression of leptin incardiovascular cells is via triplex-forming oligonucleotides (TFOs).Recent studies show that TFOs can be designed to recognize and bind topolypurine or polypirimidine regions in double-stranded helical DNA in asequence-specific manner. These recognition rules are outlined in; MaherIII, L. J., et al. (1989). Inhibition of DNA binding proteins byoligonucleotide-directed triple helix formation. Science 245, 725730;Moser, H. E., et al. (1987). Sequence-specific cleavage of doublehelical DNA by triple helix formation. Science 238, 645-650; Beal, P. A.and Dervan, P. B. (1991). Second structural motif for recognition of DNAby oligonucleotide-directed triple-helix formation. Science 251,1360-1363; Cooney, M., et al. (1988). Science 241, 456-459; and Hogan,M. E., et al., EP Publication 375408. Modifications of theoligonucleotides, such as the introduction of intercalators and backbonesubstitutions, and optimization of binding conditions (e.g., pH andcation concentration) have aided in overcoming inherent obstacles to TFOactivity such as charge repulsion and instability, and it was recentlyshown that synthetic oligonucleotides can be targeted to specificsequences (for a recent review, see Seidman, M. M. and Glazer, P. M.(2003). The potential for gene repair via triple helix formation J ClinInvest 112, 487-494.

As is described hereinabove, the present invention can be used to treatcardiovascular disorders affecting heart or vascular tissue. Thefollowing describes several options for local delivery of a leptinantagonist to tissue, for example heart and other cardiovascular tissue,specifically muscle and valve tissue.

(i) Arterial catheterization can be used to deploy a medical device suchas a mesh, a thin film, a biodegradable scaffold, a stent cover, astent, a graft assembly, a coil, a stent, a ring or a prosthetic cardiacvalve loaded with a leptin antagonist against a luminal wall of anascending aorta distal to the orifice of the coronary arteries. In caseof aneurysm at another location along the aorta, a visceral artery, orsmall tributary; the same intra-arterial approach can be used for localapplication.

(ii) An IFSD (for example, as described above, e.g., a gel) loaded witha leptin antagonist can be delivered via a balloon or needle to theaortic wall.

(iii) A composition such as a pliable non-degradable or biodegradablemesh or film loaded with the leptin antagonist that is placed in contactwith an outer surface of tissue or an organ to be treated, e.g., bysurgical delivery via open surgery or thorascopy, for example surgicallydelivered to the peri-aortic region (above the aortic root level). Incase of small aneurysm in the abdominal aorta the leptin antagonistextended release film or mesh can be applied via open surgery orminimally invasive laparoscopy.

Method of Treatment

According to an aspect of some embodiments of the invention, there isalso provided a method of treatment comprising: exposing in vivo tissueof a subject in need thereof to a pharmaceutically-effective amount ofleptin antagonist thereby providing a therapeutic effect to the tissue.For example, in some such embodiments, a composition comprising leptinantagonist is administered (e.g., by injection) directly into thetissue. In some embodiments, the exposing of the in vivo tissue to theleptin antagonist is substantially continuously for a period of not lessthan three days. In some embodiments, the period is not less than fivedays, not less than 8 days and even not less than 14 days. For example,in some such embodiments, an extended release composition comprisingleptin antagonist (e.g., a medical device impregnated with, coated withor made from a leptin antagonist is placed directly in contact with thetissue.

According to an aspect of some embodiments of the invention, there isalso provided a method of treatment comprising implanting in contactwith tissue in need thereof in the body of a subject a compositionconfigured for the in vivo release of leptin antagonist, therebyproviding a therapeutic effect to the tissue. In some embodiments, thecomposition is configured for sustained release of the leptinantagonist. In some embodiments, the implanting is intracavitaryimplanting within a fluid-filled bodily cavity of the subject. In someembodiments, by sustained release is meant that, when the composition isimplanted in vivo, leptin antagonist is released from the composition inpharmaceutically effective amounts for a period of not less than threedays, in some embodiments not less than five days, not less than 8 daysand even not less than 14 days.

The subject in need thereof is any suitable mammalian subject. In someembodiments, the subject is a non-human animal. In some embodiments, thesubject is a human.

The need is any suitable need. In some embodiments, the need is at leastone need selected from the group consisting of: attenuating a pathology;reducing the chance of developing a pathology; reducing the rate ofdevelopment a pathology; and mitigating the effect of a pathology.

The pathology is any pathology that can be treated by localadministration of leptin antagonist, and in some embodiments,substantial continuous local exposure to leptin antagonist. In someembodiments, the pathology is at least one pathology selected from thegroup consisting of:

cardiovascular disease;

remodeling of stable athersclerotic plaque into an unstable lesion(vulnerable plaque or rupture-prone plaque);

ascending aortic aneurysm, in some embodiments ascending aortic aneurysmassociated with at least one member of the group consisting ofhypertension, dyslipidermia, hypercholesterolemia, obesity, diabetesmellitus and bicuspid aortic valve (BAV);

Thoracic aortic aneurysm (e.g., to prevent rupture or dissectionthereof);

Takayasu disease (e.g., to attenuate cellular proliferative response, insome embodiments, by administration or implantation of a leptinantagonist composition to an inner or outer vessel wall in the vicinityof a vascular lesion);

Rheumatoid arteritis (e.g., to attenuate cellular proliferativeresponse, in some embodiments, by administration or implantation of aleptin antagonist composition to an inner or outer vessel wall in thevicinity of a vascular lesion);

Marfan's syndrome (by mitigation or prevention of ascending aorticaneurysms or pulmonary artery aneurysms, in some embodiments, byperivascular administration or deployment of a leptin antagonistcomposition to the outer or inner wall of the ascending aorta); giantcell arteritis; ankylosing spondylitis; inflammatory aortic aneurysm;peripheral arterial or venous aneurysms; prevention of arterialdilatation at site of anchorage of bridging stent grafts (“landingzone”) applied for EVAR (in the abdominal or thoracic aorta), visceralor peripheral arteries; prevention of myointimal hyperplasia at sites ofvascular injury; prevention of restenosis following PTA or PTCA(peripheral or cardiac balloon angioplasty); angiogenesis; cancer; andarteriovenous malformation (e.g., administration of leptin antagonistcomposition directly into a malformation or into the feeding artery).

Depending on the embodiment, any pathology, including any pathologylisted above, may be treated in accordance with the teachings herein bylocal administration of leptin antagonist. For example, in someembodiments, administration is local administration of a dose(optionally repeated) of leptin antagonist, for example by directinjection into the affected tissue or tissue proximal to the affectedtissue or with the use of a drug-eluting balloon. For example, in someembodiments, administration is local administration by a sustainedrelease composition (that releases a pharmaceutically-effective amountof leptin antagonist for a period of not less than three days) placed incontact with an outer surface of tissue (e.g., with tunica externa),inside the tissue (e.g., injection of a IFSD as described above into thetissue) or with a composition placed in contact with an inner surface ofa tissue (e.g. with tunica intima).

The tissue is any suitable tissue. In some embodiments the tissue ispart of the cardiovascular system. In some such embodiments, the tissueis selected from the group consisting of arteries, coronary arteries,ascending aorta, abdominal aorta, mitral valve, aortic valve andpulmonary valve. In some embodiments, the tissue is cardiovasculartissue with accumulated plaque, for example, an artery with accumulatedplaque. In some embodiments, the tissue is a tumor, especially acancerous tumor that grows or spreads in a process that includesangiogenesis. In some embodiments, the tissue is an arteriovenousmalformation.

In some embodiments, the leptin antagonist composition is locallyadministered during or post surgery, e.g., following carotidthrombendartrectomy or after ablation of atherosclerotic occlusion forma vessel.

In some embodiments, the leptin antagonist is locally administered bycontact to the outside of tissue to be treated, e.g., in contact withtunica externa.

In some embodiments, the leptin antagonist is locally administeredinside tissue, for example, is injected or implanted inside tissue suchas a tumor or the site of arteriovenous malformation.

In some embodiments, the leptin antagonist is locally administeredintraluminally, e.g., a leptin antagonist composition is deployed incontact with a tunica intima, inside a fluid-filled bodily cavity suchas inside the lumen of a blood vessel e.g., using an intraluminalcatheter, for example, in conjunction with a stent or prosthetic cardiacvalve.

It should be noted that in some embodiments, local administration of aleptin antagonist in accordance with the teachings herein at theascending aorta may be effective in attenuating ascending aorticaneurysms, as well as moderating left ventricular hypertrophy, and leftheart valve thickness (aortic and mitral). Administration of leptinantagonist at arterial aneurysms in other locations is anticipated toachieve a similar outcome, attenuating aneurysm expansion.

Accordingly, embodiments of the teachings herein are used to treatcardiovascular disorders such as heart valve stenosis, arterial orvenous aneurysms, or left ventricular remodeling by enabling localizedrelease of a leptin antagonist at the site of treatment.

Pharmaceutical Composition and Method of Making PharmaceuticalComposition

According to an aspect of some embodiments of the teachings herein,there is also provided a pharmaceutical composition, comprising: as anactive ingredient a leptin antagonist; and a pharmaceutically acceptablecarrier configured for in vivo sustained release of the leptinantagonist.

According to an aspect of some embodiments of the teachings herein,there is also provided a method of making a pharmaceutical composition,comprising: combining a leptin antagonist; and a pharmaceuticallyacceptable carrier configured for in vivo sustained release of theleptin antagonist.

In some embodiments, by sustained release is meant that, when thecomposition is implanted in vivo, leptin antagonist is released from thecomposition in pharmaceutically effective amounts for a period of notless than three days. in some embodiments not less than five days, notless than 8 days and even not less than 14 days.

In some embodiments, the in vivo implantation is in a human subject. Thein vivo implantation is in any suitable location. In some embodiments,the in vivo implantation is contacting an organ through a serous tissuelayer or adventitia (tunica externa) layer covering the organ, e.g., isplaced contacting a blood vessel such as the aorta from the outside ofthe blood vessel. In some embodiments, the in vivo implantation isoutside an organ directly contacting tissue of the organ (for organscovered with serous tissue, the composition is implanted underneath theserous tissue). In some embodiments, the in vivo implantation is into anorgan. In some embodiments, the implantation is from inside a hollowdefined by the organ, for example, inside a blood vessel lumencontacting the endothelium thereof.

In some embodiments, the composition is in the form of a leptinantagonist containing sheet, in some such embodiments configured to becontacted with in vivo tissue, for example, by suturing, with the use ofbiological adhesive, or pressed against the tissue, for example with thehelp of a stent or such component, e.g., the sheet is used as a stentcover for a balloon-expandable or self-expanding stent.

In some embodiments, the composition is configured to coat or besupported by an implantable medical device, e.g., is used as a coatingfor, is adsorbed or absorbed into or onto a stent (thereby constitutinga drug-eluting balloon expandable or self-expanding stent), prostheticvalve (e.g., cardiac valve), implantable spike or rod.

In some embodiments, the composition is formed into the shape of animplantable medical device, e.g., a bioresorbable stent, a bioresorbablespike or rod.

In some embodiments, the composition is injectable, e.g., is a viscousfluid or a fluid that subsequent to injection solidifies or gels, e.g.,a hydrogel.

Any suitable pharmaceutically-acceptable carrier that can be configuredfor in vivo sustained release of the leptin antagonist can be used. Insome embodiments the carrier comprises a biodegradable polymer. In somesuch embodiments, the carrier comprises a polymer selected from thegroup consisting of a hydrogel, poly glycolic acid (PGA), poly lacticco-glycolic acid (PLGA), polylactide (PLA), and poly (L-lactide) (PLLA),and combinations thereof.

The pharmaceutical composition and methods of making such a compositionare in accordance with those known in the art of pharmacology, using anysuitable method or combination of methods as known in the art such asdescribed in “Remington's Pharmaceutical Sciences,” Mack Publishing Co.,Easton, Pa., latest edition, which is incorporated herein by reference.Such methods include conventional mixing, curing, polymerizing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing. Pharmaceutical compositionsmay be formulated in conventional manner using one or morephysiologically acceptable carriers, diluents, excipients or auxiliarieswhich facilitate processing of the leptin antagonist into apharmaceutical composition.

Use of Leptin Antagonist

According to an aspect of some embodiments of the teachings herein,there is also provided a use of a leptin antagonist according to theteachings herein for the localized treatment of tissue of a livingorganism, comprising, implanting a composition comprising a leptinantagonist in vivo to contact tissue in need thereof so that the tissueis exposed to a pharmaceutically effective amount of leptin antagonist,thereby providing a therapeutic effect to the tissue. In someembodiments, the composition and the implanting is such that the tissueis exposed to a pharmaceutically effective amount of leptin antagonistsubstantially continuously for a period of at least three days. In someembodiments, the period is not less than five days, not less than 8 daysand even not less than 14 days.

In some embodiments, an above administration is periodically repeated.For example, in some embodiments, administration of leptin antagonist isrepeated. For example, in some embodiments, administration of leptinantagonist is repeated after at least a period, the period selected fromthe group consisting of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2months and 3 months.

Treatment of Athersclerotic Plaque

It is known that stable athersclerotic plaque accumulates in the innerwalls of mammalian arteries. In some instances, the stable plaque orrupture-prone plaque. The unstable lesion may disintegrate, formingemboli.

An aspect of the teachings herein is based on the Inventor's discoverythat locally synthesized leptin within the carotid atheroscleroticplaque, which characterize unstable plaques (rupture prone), correlateswith brain emboli. Therefore, the Inventor believes that a leptinantagonist administered to a stable athersclerotic plaque should reducethe rate and/or incidence of the conversion of a stable atherscleroticplaque to an unstable lesion.

According to an aspect of some embodiments of the teachings herein,there is provided a method for treatment of athersclerotic plaque,comprising: administering a pharmaceutically-effective amount of aleptin antagonist to athersclerotic plaque accumulated in the innerwalls of an artery, thereby at least one of: (a) reducing the rate and(b) reducing the incidence, of conversion of a stable atherscleroticplaque to an unstable lesion.

Administration of the leptin antagonist is any suitable administration.In some embodiments, the administration is by sustained-release of theleptin antagonist directly to an inner wall in which plaque isaccumulated, from a leptin antagonist containing composition. In someembodiments, such sustained release is substantial continuous release ofa pharmaceutically-effective amount of leptin antagonist for a period ofnot less than three days, not less than 5 days, not less than 8 days andeven not less than 14 days. In some such embodiments, the composition isin direct contact with the surface of the plaque to be treated. In someembodiments, the composition is in contact with the inner walls of ablood vessel with accumulated plaque.

Surgical Connecting Devices

It is known that trauma to a blood vessel may lead to myointimalhyperplasia (MIH). One type of trauma that may cause MIH is caused bysurgical connecting devices such as surgical staples and suture threadsthat are applied to blood vessels, for example, during surgery forexample surgical anastomosis. It has been found that in some instances,local administration of leptin antagonist to such wounds may be able tomitigate or prevent MIH.

Thus, according to an aspect of some embodiments of the invention, thereis also provided a surgical connecting device, comprising: a soliddevice body made of a material; and functionally associated with thedevice body, a pharmaceutically-effective amount of leptin antagonist.In some embodiments, the device body is in the form selected from thegroup consisting of surgical suture thread and a surgical staple.

An embodiment of a suture thread 10 and of a surgical staple 12 inaccordance with the teachings herein are schematically depicted in FIG.27.

In some embodiments, the device body, e.g., of thread 10 or staple 12,is absorbable (i.e., bioresorbable).

The device body, e.g., of thread 10 or staple 12, is made of anysuitable material. In some embodiments, the device body is made of amaterial comprises a polymer selected from the group consisting of polyglycolic acid (PGA), poly lactic co-glycolic acid (PLGA), polylactide(PLA), and poly (L-lactide) (PLLA), and combinations thereof.

The leptin antagonist is functionally associated with the device body,e.g., of thread 10 or staple 12, in any suitable manner.

In some embodiments, the functional association of the leptin antagonistwith the device body is that a composition comprising the leptinantagonist coats the device body. For example, in some such embodimentsthread 10 or staple 12 are made of PGA, and coated with a coating ofPLGA that includes a pharmaceutically effective amount of leptinantagonist.

In some embodiments, the functional association of the leptin antagonistwith the device body is that a composition comprising the leptinantagonist impregantes the device body. For example, in some suchembodiments thread 10 is made of silk and impregnated with a compositionthat includes a pharmaceutically effective amount of leptin antagonist.

In some embodiments, the functional association of the leptin antagonistwith the device body is that the material from which the device body ismade is a composition comprising the leptin antagonist. For example, insome such embodiments thread 10 or staple 12 are made of PGA thatincludes a pharmaceutically effective amount of leptin antagonist.

Administration of Leptin Antagonist in Fluid-Filled Cavities

As noted above, the present inventor has found that in vivo implantationinside a fluid-filled cavity of the body (for example of the cardiovascular system such as blood vessels or cardiac chambers) can have adesirable pharmaceutical effect on tissue in proximity of the implantedcomposition with limited or no substantial side-effects:

Method of Treatment in a Fluid-Filled Bodily Cavity

Thus, according to an aspect of some embodiments of the invention, thereis also provided a method of treating a condition in a subject in needthereof, the method comprising administering intracavitarily to innerwalls of a fluid-filled bodily cavity of the subject a compositioncomprising a leptin antagonist. In some embodiments of the method, thesubject is human. In some embodiments of the method, the subject is anon-human animal.

In some embodiments of the method, the intracavitary administrationexposes in vivo tissue to a pharmaceutically-effective amount of theleptin antagonist and thereby provides a therapeutic effect to the invivo tissue.

In some embodiments of the method, the in vivo tissue comprises tissueof the inner walls of the cavity (e.g., tunica intima).

In some embodiments of the method, the administration is localadministration. In some such embodiments, the in vivo tissue exposed tothe pharmaceutically-effective amount of the leptin antagonist isexclusively tissue in physical proximity to the administeredcomposition. In some such embodiments, the in vivo tissue exposed to thepharmaceutically-effective amount of the leptin antagonist isexclusively tissue in physical contact with the administeredcomposition.

In some embodiments of the method, exposing of the in vivo tissue to apharmaceutically-effective amount of the leptin antagonist issubstantially continuously for a period of at least three days, at leastfive days, at least eight days and in some embodiments, even at leastfourteen days.

In some embodiments of the method, the composition is asustained-release composition, configured for sustained release of apharmaceutically-effective amount of the leptin antagonist when locatedinside a bodily cavity. In some embodiments, the sustained releasecomprises release of a pharmaceutically-effective amount of the leptinantagonist over a period of at least three days, at least five days, atleast eight days and in some embodiments, even at least fourteen days.

In some embodiments of the method, the intracavitary administeringcomprises implantation of the composition within the cavity in contactwith the inner walls of the cavity.

In some embodiments of the method, the intracavitary administeringcomprises deploying an intracavitarily-implantable medical device in thecavity. In some such embodiments, the medical device is deployed incontact with the inner walls of the cavity. In some such embodiments,the leptin antagonist is functionally associated with the deployedintracavitarily-implantable medical device. In some preferred suchembodiments, the leptin antagonist is functionally associated with aportion of the deployed intracavitarily-implantable medical device thatcontacts bodily tissue when the medical device is deployed. In some suchembodiments, the intracavitarily implantable medical device is selectedfrom the group consisting of: a stent cover, a graft assembly, a coil(e.g., aneurysm coil), a stent (e.g., expandable stent, self-expandingstent, covered stent, partially covered stent, not covered stent), aring (e.g, a graft anchor), a suture, a staple and a prosthetic cardiacvalve. Although the teachings herein are applicable to any prostheticcardiac valve, the teachings are particularly advantageous forimplementing with catheter-deployed prosthetic cardiac valves (e.g.,TAMVI, TAVI): since these valves are typically held in place withoutsutures so that myointimal hyperplasia that potentially develops as aresult of trauma caused during deployment may lead to leakage.

Composition for Treatment in a Fluid-Filled Bodily Cavity

According to an aspect of some embodiments of the invention, there isalso provided a composition comprising: a leptin antagonist for use intreating a condition, wherein the composition is configured forintracavitary administration to inner walls of a fluid-filled bodilycavity of a subject. In some embodiments of the composition, the subjectis human. In some embodiments of the composition, the subject is annon-human animal.

In some embodiments, the intracavitary administration of the compositionexposes in vivo tissue to a pharmaceutically-effective amount of theleptin antagonist, thereby providing a therapeutic effect to the in vivotissue. In some embodiments, such in vivo tissue comprises tissue ofinner walls of a the cavity.

In some embodiments, the administration is local administration. In somesuch embodiments, the in vivo tissue exposed to thepharmaceutically-effective amount of the leptin antagonist isexclusively tissue in physical proximity to the administeredcomposition. In some such embodiments, the in vivo tissue exposed to thepharmaceutically-effective amount of the leptin antagonist isexclusively tissue in physical contact with the administeredcomposition.

In some embodiments of the composition, exposing of the in vivo tissueto a pharmaceutically-effective amount of the leptin antagonist issubstantially continuously for a period of at least three days, at leastfive days, at least eight days and in some embodiments, even at leastfourteen days.

In some embodiments, the composition is a sustained-release composition,configured for sustained release of a pharmaceutically-effective amountof the leptin antagonist when located inside a bodily cavity. In someembodiments, the sustained release comprises release of apharmaceutically-effective amount of the leptin antagonist over a periodof at least three days, at least five days, at least eight days and insome embodiments, even at least fourteen days.

In some embodiments of the composition, the configuration forintracavitary administration comprises configuration for implantation ofthe composition within the cavity in contact with the inner walls of thecavity.

In some embodiments of the composition, the configuration forintracavitary administration comprises configuration for deploying withan intracavitarily-implantable medical device in the cavity. In somesuch embodiments, the medical device is configured for deployment incontact with the inner walls of the cavity. In some such embodiments,the leptin antagonist is functionally associated with theintracavitarily-implantable medical device. In some preferred suchembodiments, the leptin antagonist is functionally associated with aportion of the intracavitarily-implantable medical device that contactsbodily tissue when the medical device is deployed. In some suchembodiments, the intracavitarily implantable medical device is selectedfrom the group consisting of: a stent cover, a graft assembly, a coil(e.g., aneurysm coil), a stent (e.g., expandable stent, self-expandingstent, covered stent, partially covered stent, not covered stent), aring (e.g, a graft anchor), a suture, a staple and a prosthetic cardiacvalve. As noted above, although the teachings herein are applicable toany prosthetic cardiac valve. As noted above, although the teachingsherein are applicable to any prosthetic cardiac valve, the teachings areparticularly advantageous for implementing with catheter-deployedprosthetic cardiac valves (e.g., TAMVI, TAVI).

In some embodiments of the method or composition, the condition is apathological cardiovascular condition. In some embodiments of the methodor composition, the condition is a cardiovascular condition selectedfrom the group consisting of atherosclerosis, valve stenosis, aneurysms,vessel response to vascular injury, and cardiomyopathy. In someembodiments of the method or composition, the administration of theleptin antagonist is to athersclerotic plaque accumulated in the innerwalls of an artery, thereby at least one of: (a) reducing the rate and(b) reducing the incidence, of conversion of a stable atherscleroticplaque to an unstable lesion. In some embodiments of the method orcomposition, the administration of the leptin antagonist is to bodilytissue in order to prevent or mitigate the development of myointimalhyperplasia, for example, myointimal hyperplasia that potentiallydevelops as a result of trauma caused during deployment of a medicaldevice in a the body of a living subject.

In some embodiments of the method or composition, the compositionconstitutes a coating of an intracavitarily implantable medical device.In some embodiments of the method or composition, the composition isimpregnated in an intracavitarily impantable medical device. In somesuch embodiments of the method or composition, the compositioncomprises, in addition to the leptin antagonist, a polymer selected fromthe group consisting of a hydrogel, poly glycolic acid (PGA), polylactic co-glycolic acid (PLGA), polylactide (PLA), and poly (L-lactide)(PLLA), and combinations thereof.

In some embodiments of the method or composition, the composition is inthe form selected from the group consisting of a sheet and a tubeconstituting a portion of an intracavitarily implantable medical devicecomprising the leptin antagonist. In some such embodiments of the methodor composition, the composition comprises, in addition to the leptinantagonist, a polymer selected form the group consisting of of ahydrogel, poly glycolic acid (PGA), poly lactic co-glycolic acid (PLGA),polylactide (PLA), and poly (L-lactide) (PLLA), and combinationsthereof. In some such embodiments of the method or composition, theintracavitarily implantable medical device comprises a stent and thecomposition constitutes a stent cover (e.g., a partial or complete stentcover, a balloon-expandable or a self-expanding stent). In some suchembodiments of the method or composition, the intracavitarilyimplantable medical device comprises a graft-assembly (e.g., astent-graft or ring-graft assembly) and the composition constitutes agraft portion thereof. For example, some embodiments are configured tofunction as a stent-graft assembly for treatment of AAA, like theEndurant® II by Medtronic (Dublin, Ireland).

In some embodiments of the method or composition, the compositionconstitutes at least a portion of an intracavitarily implantable medicaldevice, and in some embodiments, the composition constitutessubstantially an entire intracavitarily implantable medical device. Asnoted above, in some such embodiments of the method or composition, theintracavitarily implantable medical device is selected from the groupconsisting of a stent cover, a graft assembly, a coil (e.g., aneurysmcoil), a stent (e.g., expandable stent, self-expanding stent, coveredstent, partially covered stent, not covered stent, a ring (e.g, a graftanchor), a suture, a staple and a prosthetic cardiac valve. In some suchembodiments of the method or composition, the composition comprises, inaddition to the leptin antagonist, a polymer selected from the groupconsisting of a hydrogel, poly glycolic acid (PGA), poly lacticco-glycolic acid (PLGA), polylactide (PLA), and poly (L-lactide) (PLLA),and combinations thereof.

Medical Device for Treatment in a Fluid-Filled Bodily Cavity

According to an aspect of some embodiments of the invention, there isalso provided an intracavitarily-implantable medical device, comprising:

at least one solid functional device part configured for deploying thedevice in a fluid-filled bodily cavity of a subject; and

functionally associated with at least one the device component, a leptinantagonist.

In some embodiments of the medical device part as a component of apharmaceutical composition comprising the leptin antagonist.

In some embodiments of the medical device, the pharmaceuticalcomposition is a sustained-release composition, configured for sustainedrelease of a pharmaceutically-effective amount of the leptin antagonistwhen located inside a bodily cavity.

In some embodiments of the medical device, sustained release comprisesrelease of a pharmaceutically-effective amount of the leptin antagonistover a period of at least three days, at least five days, at least eightdays and in some embodiments, even at least fourteen days.

In some embodiments of the medical device, thepharmaceutically-acceptable carrier further comprises a polymer selectedfrom the group consisting of a hydrogel, poly glycolic acid (PGA), polylactic co-glycolic acid (PLGA), polylactide (PLA), and poly (L-lactide)(PLLA), and combinations thereof.

In some embodiments of the medical device, the functional association isat least one device component having a coating comprising thepharmaceutical composition. In some embodiments of the medical device,the functional association is at least one device component beingimpregnated with the pharmaceutical composition. In some embodiments ofthe medical device, the functional association is at least one devicecomponent being fashioned of the pharmaceutical composition.

In some embodiments of the medical device, the medical device isselected from the group consisting of a stent cover, a graft assembly, acoil (e.g., aneurysm coil), a stent (e.g., expandable stent,self-expanding stent, covered stent, partially covered stent, notcovered stent), a ring (e.g, a graft anchor), a suture, a staple and aprosthetic cardiac valve (as noted above, preferably catheter-deployedprosthetic cardiac valves.

In some embodiments of the method, composition and device, thefluid-filled bodily cavity is a bodily cavity of the cardiovascularsystem. In some such embodiments, the cavity is selected from the groupconsisting of a cardiac chamber, an artery and a vein. In some suchembodiments, the cardiac chamber is selected from the group consistingof left ventricle, right ventricle, left atrium and right atrium. Insome such embodiments, the artery is selected from the group consistingof a systemic artery, a cardiac artery and a pulmonary artery. In somesuch embodiments, the systemic artery is an aorta, for example selectedfrom the group consisting of an ascending aorta, aortic arch, descendingaorta and an abdominal aorta.

In FIG. 28, an aneurysm coil 14 according to the teachings herein isschematically depicted. Aneurysm coil 14 is substantially similar toknown aneurysm coils except by being functionally associated with apharmaceutically-effective amount of leptin antagonist, is made insubstantially the same way, and is used in substantially the same way.Depending on the embodiment, a given aneurysm coil 14 may include one ormore of the additional features detailed hereinabove. For example, insome embodiments, coil 14 is made of plantinum, and coated with acoating of a hydrogel that includes a pharmaceutically effective amountof leptin antagonist. For example, in some embodiments, coil 14 is madeof a material such as platinum or a polymer textured with micrometerdimension features such as valleys and pores, where inside the featuresis held a composition (e.g., a gel such as of Example 11) that comprisesleptin antagonist. For example, in some embodiments, coil 14 is made ofa composition comprising the leptin antagonist, e.g., is made of PGAthat includes a pharmaceutically effective amount of leptin antagonist.

In FIG. 28, a prosthetic cardiac valve 16 according to the teachingsherein is schematically depicted. Prosthetic cardiac valve 16 issubstantially similar to known prosthetic cardiac valves except by beingfunctionally associated with a pharmaceutically-effective amount ofleptin antagonist, is made in substantially the same way, and is used insubstantially the same way. Specifically, prosthetic cardiac valve 16has at least one component that is functionally associated with apharmaceutically-effective amount of leptin antagonist. Depending on theembodiment, a given prosthetic cardiac valve 16 according to theteachings herein may include one or more components, each having one ormore of the additional features detailed hereinabove. For example, insome embodiments, the retainer ring of prosthetic cardiac valve 16 ismade of a cobalt chromium ring with a polyester cloth cover, the ringand cloth cover both coated with a coating of a hydrogel that includes apharmaceutically effective amount of leptin antagonist. For example, insome embodiments, the leaflets of prosthetic cardiac valve 16 are madeof a material such as porcine or bovine tissue (e.g., cardiac leaflets,pericardium) that has been soaked in and is therefore impregnated with acomposition (e.g., a gel such as of Example 11) that comprises leptinantagonist.

In FIG. 28, a graft assembly 18 according to the teachings herein isschematically depicted. Graft assembly 18 is a ring-graft assemblysuitable for treatment of abdominal aorta aneurysms and includes aflexible graft 20 that defines a conduit for blood flow and threeexpandable anchoring rings 22 as graft anchors. Each anchoring ring 22is a radially expandable device that is substantially a single 360° ringof material. As known in the art, some embodiments of graft assembliesare stent-graft assemblies where one or more of the anchors are radiallyexpandable stents, that are longer in the axial direction and/ordescribe more than a 360° degree rotation and/or comprise more than asingle ring of material. Stents are preferred as anchors as thesetypically also provide support for the vessel in which deployed andprovide greater anchoring of the graft to a vessel in which deployed.Graft assembly 18 is substantially similar to known graft assembliesexcept by being functionally associated with apharmaceutically-effective amount of leptin antagonist, is made insubstantially the same way, and is used in substantially the same way.Specifically, graft assembly 18 has at least one component that isfunctionally associated with a pharmaceutically-effective amount ofleptin antagonist. Depending on the embodiment, a given graft assembly18 according to the teachings herein may include one or more components,each having one or more of the additional features detailed hereinabove.

For example, in some embodiments, graft 20 is substantially a tube madeof a high-density multifilament polyester cloth coated with a coating ofa hydrogel that includes a pharmaceutically effective amount of leptinantagonist. In some embodiments, the coating is on the entire outersurface of graft 20. In some embodiments, the coating is on the outersurface of the termini of the three legs of graft 20 (e.g., a 5 cmlength from each terminus.

For example, in some embodiments, graft 20 is made of a high-densitymultifilament polyester cloth coat that has been soaked in and istherefor impregnated with a composition (e.g., a gel such as of Example11) that comprises leptin antagonist. In some embodiments, the entiregraft 20 is impregnated with leptin antagonist composition. In someembodiments, only the termini of the three legs of graft 20 (e.g., a 5cm length from each terminus) is impregnated with leptin antagonistcomposition.

For example, in some embodiments, rings 22 are made of nitinol, andcoated with a coating of a hydrogel that includes a pharmaceuticallyeffective amount of leptin antagonist. For example, in some embodiments,rings 22 are made of a material such as nitinol textured with micrometerdimension features such as valleys and pores, where inside the featuresis held a composition (e.g., a gel such as of Example 11) that comprisesleptin antagonist.

In FIG. 29, stents 24, 26 and 28 according to the teachings herein areschematically depicted. Stents 24, 26 and 28 are all elongated, tubular,outwardly radially-expandable frameworks that are known in the art.Stent 24 is a coverless stent without a cover. Stent 26 is apartially-covered stent with a partial cover 30. Partial cover 30 is asheet secured to the framework of stent 26 in the usual way, e.g., withsutures. Stent 28 is a covered stent with a full cover 32. Full cover 30is a tube secured to the framework of stent 28 in the usual way, e.g.,with sutures or by tension.

Stents 24, 26 and 28 are substantially similar to known stents except bybeing functionally associated with a pharmaceutically-effective amountof leptin antagonist, are made in substantially the same way, and areused in substantially the same way. Specifically, each one of stents 24,26 and 28 has at least one component that is functionally associatedwith a pharmaceutically-effective amount of leptin antagonist. Dependingon the embodiment, a given stent 24, 26 and 28 according to theteachings herein may include one or more components, each having one ormore of the additional features detailed hereinabove. Embodiments of anyone of stents 24, 26 and 28 are self-expanding stents. Embodiments ofany one of stents 24, 26 and 28 are balloon-expandable stents.

For example, in some embodiments, a cover 30 or a cover 32 is made of ahigh-density multifilament polyester cloth coated with a coating of ahydrogel that includes a pharmaceutically effective amount of leptinantagonist, typically on the outer surface of the cover.

For example, in some embodiments, a cover 30 or a cover 32 is made of ahigh-density multifilament polyester cloth coated that has been soakedin and is therefore impregnated with a composition (e.g., a gel such asof Example 11) that comprises leptin antagonist.

For example, in some embodiments, a cover 30 or a cover 32 is made of amaterial that is a composition comprising leptin antagonist, e.g., aPLGA sheet of example 4.

For example, in some embodiments, a framework of any one of stents 24,26 and 28 is made of cobalt chrominum coated with a coating of ahydrogel that includes a pharmaceutically effective amount of leptinantagonist.

For example, in some embodiments, a framework of any one of stents 24,26 and 28 is made of a material such as nitinol or a polymer texturedwith micrometer dimension features such as valleys and pores, whereinside the features is held a composition (e.g., a gel such as ofExample 11) that comprises leptin antagonist.

For example, in some embodiments, a framework of any one of stents 24,26 and 28 is made of a material that is a composition comprising leptinantagonist, e.g., PLA or PLLA comprising a pharmaceutically-effectiveamount of leptin antagonist.

Suitable Leptin Antagonists

Any suitable leptin antagonist may be used in implementing any specificaspect or embodiment of the teachings herein. In some embodiments, asingle leptin antagonist is used. In some embodiments, two or moreleptin antagonists are used simultaneously or concurrently.

Various leptin antagonists suitable for implementing the teachingsherein have been described in detail hereinabove.

In some embodiments, the leptin antagonist comprises a polypeptideportion.

In some embodiments, the leptin antagonist is a polypeptide.

Preferred leptin antagonists include all of the leptin antagonistslisted and taught in U.S. Pat. No. 7,307,142 (SEQ ID NOs 3-35) and U.S.Pat. No. 8,969,292 (SEQ ID NOs 36-47), which are both included byreference as if fully set-forth herein. In some embodiments, the leptinantagonist is selected from the group consisting of:

-   -   a leptin antagonist consisting of: (a) a mammalian leptin        polypeptide in which the LDFI (SEQ ID NO:33 in U.S. 7,307,142 or        SEQ ID NO 35 in the present application) hydrophobic binding        site at the positions corresponding to positions 39-42 of the        wild-type human leptin, is modified such that from two to four        amino acid residues of the hydrophobic binding site are        substituted with different amino acid residues such that the        site becomes less hydrophobic, the modified, mammalian leptin        polypeptide being a leptin antagonist; (b) a fragment of the        modified mammalian leptin polypeptide of (a) comprising the        altered hydrophobic binding site, wherein the fragment is itself        a leptin antagonist; (c) a fragment of (b)    -   a synthetic leptin antagonist comprising: (d) a full length        modified mammalian leptin polypeptide in which: (i) the LDFI        hydrophobic binding site at the position corresponding to        positions 39-42 of the wild-type human leptin is modified such        that from two to four amino acid residues of the hydrophobic        binding site are substituted with different amino acid residues        such that the site becomes less hydrophobic; and    -   (ii) the aspartic acid at the position corresponding to position        23 of the wild-type human leptin D23) is substituted with an        amino acid residue selected from the group consisting of        glycine, alanine, leucine, lysine, arginine, phenylalanine,        tryptophan and histidine, or threonine at the position        corresponding to position 12 of the wild-type human leptin (T12)        is substituted with a different amino acid residue that is        hydrophobic; (e) a synthetic leptin antagonist consisting of a        polypeptide having the amino acid sequence of SEQ ID NO: 1 of        U.S. Pat. No. 8,969,292 or SEQ ID NO 36 of the present        application;        (f) a modified mammalian leptin polypeptide in which: (i) the        LDFI hydrophobic binding site at the position corresponding to 5        positions 39-42 of the wild-type human leptin is modified such        that from two to four amino acid residues of the hydrophobic        binding site are substituted with different amino acid residues        such that the site becomes less hydrophobic, the modified        mammalian leptin polypeptide being a leptin antagonist; and    -   the aspartic acid at the position corresponding to position 23        of the wild-0 type human leptin (D23) is substituted with a        different amino acid residue that is not negatively charged or        the threonine at the position corresponding to position 12 of        the wild-type human leptin (T12) is substituted with a different        amino acid residue that is hydrophobic;    -   (g) a fragment of the modified mammalian leptin polypeptide of        (f), in which D235 is substituted with a different amino acid        residue that is not negatively charged or T12 is substituted        with a different amino acid residue that is hydrophobic, wherein        the fragment is itself a leptin antagonist;    -   a synthetic leptin agonist comprising: (h) a modified mammalian        leptin polypeptide in which D23 is substituted with a different        amino acid residue that is not negatively charged or T12 is        substituted with a different amino acid residue that is        hydrophobic;    -   (j) a fragment of the modified mammalian leptin polypeptide of        (h), in which D23 is substituted with a different amino acid        residue that is not negatively charged or T12 is substituted        with a different amino acid residue that is hydrophobic, wherein        the fragment is itself a leptin agonist;    -   (k) any one of the above wherein the mammalian leptin is        selected from the group consisting of human, ovine and murine;    -   (l) any one of (a), (b), (c), (d), (e), (f), (g), (h), (j)        and (k) in pegylated form; and    -   a pharmaceutically acceptable salt of any one of (a)-(l).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. In case of conflict, thespecification, including definitions, will take precedence.

As used herein, intracavitary relates to within an organ or body cavity.

Art that provides enabling support for the teachings herein, and thatmay also be useful in understanding the background and the inventiveaspects of the teachings herein includes U.S. Pat. Nos. 7,307,142;8,969,292; “leptin Locally Synthesized in Carotid AtheroscleroticPlaques Could be Associated With Lesion Instability and Cerebral Emboli”by Schneiderman J et al in J Am Heart Assoc. 2012, 1:e001727 doi:10.1161/JAHA, 112.001727 and “Locally Applied leptin Induces RegionalAortic Wall Degeneration Preceding Aneurysm Formation in ApolipoproteinE—Deficient Mice” by Tao M et al in Arterioscler Thromb Vase Biol.2013;33:311-320, all four which are included by reference (together withany published supplemental materials) as if fully set-forth herein.

As used herein, the terms “comprising”, “including”, “having” andgrammatical variants thereof are to be taken as specifying the statedfeatures, integers, steps or components but do not preclude the additionof one or more additional features, integers, steps, components orgroups thereof. These terms encompass the terms “consisting of” and“consisting essentially of”.

As used herein, the indefinite articles “a” and “an” mean “at least one”or “one or more” unless the context clearly dictates otherwise.

As used herein, when a numerical value is preceded by the term “about”,the term “about” is intended to indicate +/−10%.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Example 1 Localized Leptin Synthesis in a Mouse Model

A novel mouse model was used to simulate local leptin synthesis in theascending aorta in order to assess the effect of leptin on aorticremodeling and heart structure and function.

Materials and Methods

A slow release leptin film (FIG. 1b ) made of polylactic co-glycolicacid (PLGA) matrix (1×1.5 mm), and containing either 2 μg leptin or noprotein (control) was applied to the anterior surface of the proximalascending aorta (FIG. 4).

The leptin slow-release film was manufactured by impregnating a polylactic-co-glycolic acid (PLGA) film with leptin. One gram of PLGA 6535polymer (D,L-lactide:glycolide: 65:35, Mw=45,000-75,000 Da; Lakeshore,Biomaterials, Birmingham, Ala. USA) was dissolved in 10 mL MgCl₂ (FisherScientific, Loughborough, UK). Sodium chloride (10 mg in 0.2 mLdistilled water) and 25 μL ethylene glycol (Sigma-Aldrich, St. Louis,Mo., USA) were added to the polymeric solution and sonicated for 20seconds. Mouse leptin powder (1 mg; #L3772; Sigma-Aldrich, St. Louis,Mo., USA) was suspended in 2 mL of the polymeric solution, followed bycasting on a flat surface of Teflon molds to create a flat film. Filmswere dried in a hood for 48 hours, and then subjected to high vacuum for12 hours to extract any residual solvent. Control (placebo) films werefabricated in the same way without the addition of leptin. Thecalculated amount of leptin per 1×1.5-mm film used currently forimplantation in each mouse was 2 μg.

Another option of leptin application for local slow-release has been agel composed of two liquid materials which gel (solidify) upon mixing atthe time of infection. These are a modified carboxymethyl cellulose withadipic dihydrazide (CMC-ADH) and an oxidized dextrane in DDW (DX-COH).Methylene blue dye (0.5%) was also added to the DX-COH solution to makethe resulting gel more visible. Leptin (sigma, L3772, St. Louis, Mo.,USA) was added to the gel by an emulsion technique

Serum leptin levels were determined in ApoE^(−/−) mice after receiving20 μg mouse leptin via peri-aortic application (in another experiment,Tao et al. ATVB 2013). Blood was sample on days 0, 7, 14, and 21, andleptinanalyzeded by ELISA assay (Quantikine Mouse Lep Kit, R&D Systems,Minneapolis, Minn., USA): Day 0-3.5 ng/mL; day 7—leptin 8.0 ng/mL,placebo 9.2 ng/mL; day 14—leptin 12.0 ng/mL, placebo 14.5 ng/mL; and day21—leptin 12.25 ng/mL, placebo 12.5 ng/mL (FIG. 6). Notably, thesevalues fell within the normal range of plasma leptin in ApoE−/− micereceiving Western diet (means 5.1±1.4 to 17±3.4 ng/mL). It should alsobe emphasized that circulating leptin levels are known to increase withage, as also observed in our series.

This unique mouse model was utilized to perform two experiments: Mice inexperiment 1 were fed postoperatively with high fat diet (HFD), and werefollowed up for 45 days. In experiment 2 mice received normal chow for30 or 60 days. Mouse weight and blood pressure (BP) were assessedweekly. All mice recovered from surgery uneventfully.

Results

In both mouse model experiments, leptin or control treated mice gainedweight equally during the follow up period, suggesting no systemicleptin effect. Systolic BP measured weekly in mice of experiment 2 was100 mmHg throughout the first 4 weeks, and increased to 120 mmHg by week6 in both leptin treated and control mice. Based on two separateexperiments, both HFD and normal chow feeding yielded in general similarresults.

The following data report the results of experiment 2 (normal chowfeeding).

Echocardiography of the ascending aorta at 2 mm distal to the aorticvalve level revealed an increase in aortic diameter at peak systole inleptin treated mice vs controls (p=0.08, FIG. 6; Exp. 1 using HFDyielded P<0.003). That same aortic diameter in systole vs. diastole, inleptin compared to control treated mice. There was no significantdifference in diameter further distally on the ascending aorta. Notably,the aortic valve annulus did not dilate in response to local leptinapplication. Histological analysis of the ascending aorta revealedfeatures of medial degeneration at the site of leptin application,including fragmentation of the elastic lamellas, as demonstrated byelastica van Giesen staining, and depletion of αSMA in the media (FIG.7). These structural changes likely underlie local stiffening anddilatation in the proximal ascending aorta.

Echocardiography (final vs. pre-operative) revealed a concentricremodeling of the left ventricle, with hypertrophy of all LV walls(p<0.001). Wall thickening was most pronounced in diastole (p=0.002,FIG. 8). Left ventricular diameter was increased in both systole anddiastole (p=0.08, p=0.02, respectively, FIG. 9), leading to a reductionin the LV fractional area change (FAC, p=0.07, FIG. 10).

Local leptin application at the proximal ascending aorta promotedthickening of the mitral and aortic valve leaflets (p=0.01, p<0.001accordingly, FIG. 11). Mitral leaflets were diffusely thickened, whileaortic valve leaflets displayed thickening in their free edge, composedmostly of ECM and stromal cells. These proliferating cells are assumedanalogous to human valvular interstitial cells (VICs). A few stromalcells within these lesions were positive for αSMA and TGFβ as shown byIHC staining (in analyzed aortic valve leaflets), suggesting VICsactivation (FIG. 11). A trend was observed for increased VICproliferation through Ki67 IHC in leptin treated mice. However, the lackof statistical significance implies that most leaflet hyperplasia tookplace at an earlier time.

Increased peak systolic velocity (PSV), as measured at the aortic valvein leptin treated vs control mice was short of statistical significance.However, PSV was significantly augmented in postoperative HFD fedanimals.

These experiments reveal that available leptin in the proximal ascendingaorta induces local aortic stiffening and dilation. The resultingchanges in local hemodynamics likely drive remodeling of the leftventricle, including LV wall hypertrophy and valve thickening throughthe aorto-ventricular coupling axis.

Example 2 Local Leptin Antagonism in an Ang II Mouse Model

Angiotensin II (AngII) is the key hormone of the renin-angiotensinsystem, underlying hypertension and cardiovascular remodeling (Renna etal. Pathophysiology of vascular remodeling in hypertension. Int JHypertens. 2013;2013: 808353). The phenotypes induced by local leptinapplication described in Example 1 are reminiscent of AngII inducedaortic-ventricular (coupling) remodeling, suggesting that leptinmediates these processes. As such, a leptin antagonist was deliveredlocally to the ascending aorta in order to assess the effects of leptindown-regulation on AngII induced local aortic remodeling, andaortic-ventricular remodeling in mice.

Materials and Methods

An osmotic mini-pump, delivering AngII at a rate of 1000 ng/kg/min wasimplanted subcutaneously in the back of the neck of 14 week oldApoE^(−/−) mice. Each mouse also underwent left mini-thoracotomy forapplication of a slow release miniature PLGA film (1×1.5 mm) containingeither 5 μg leptin antagonist (LA), or PLGA matrix devoid of protein(control). The slow release film was deploye on the surface of theproximal ascending aorta at the position described in Experiment 1. Micewere euthanized 4 weeks following surgery. As expected, blood pressureassessed in both Ang II treated groups after one week was increased byapproximate 20% (125 mmHg mean systolic), and was sustained throughoutthe follow up (FIG. 12). Weight gain pattern was similar in both groups,indicating no systemic effects related to the leptin antagonist (FIG.13).

Results

to assess the impact of AngII alone versus AngII plus leptin antagoniston mouse longevity, mortality data from the present experiment werecombined with data from a previous experiment, which included a similarcohort of ApoE^(−/−) mice exposed to AngII, in same dose and duration(Tao M, et al. ATVB 2013). Collectively, a 34% mortality (referred topremature death, prior to the completion of the experiment) was observedin mice treated with AngII (either Ang II alone or Ang II with controlfilm applied on the ascending aorta). Death was related to thoracic(28%) or abdominal (6%) aortic aneurysm rupture. Notably, mice treatedwith AngII that received also LA were protected form thoracic aneurysmrupture (p=0.03, p=0.005, respectively, FIG. 15). However, these datadid not suggest moderation of increased aortic stiffness by LAapplication.

Histological analysis revealed medial degeneration in both groups thatwere treated with AngII. Nevertheless, additional LA applicationresulted in less fragmentation of the elastic lamellas and fewer sitesof αSMA depletion in the aortic media (FIG. 16). Notably, amongst micereceiving AngII, medial degeneration was rather diffused throughout theaorta. This was in sharp contrast to the effects of local leptinapplication, which exhibited medial degeneration within the segment incontact with the leptin film alone.

Immunohistochemical analysis for leptin antigen revealed a weakexpression in medial SMCs, and a strong signal within foam cells ofaortic luminal atherosclerotic plaques (FIG. 17).

Mice treated with LA presented less thickening of the left ventricularwall, particularly in diastole (p<0.01, FIG. 18). Left ventriculardiameter increased similarly in both groups in diastole however, LAtreatment attenuated the increase in LV diameter during systole (p=0.05,FIG. 19). As anticipated, and corresponding to these results, a decreasein FAC in mice co-treated with AngII and LA, was observed, while micetreated by AngII alone exhibited a decrease in fractional area change byover 15% (p=0.03, FIG. 20). Moreover, LV diameter which increased inresponse to Ang II treatment, was preserved within the baseline(pre-AngII treatment) range in the LA treated mice (P<0.05).

Peak systolic velocity was decreased in Ang II treated mice that alsoreceived LA application, vs. AngII alone (p=0.03, FIG. 21). Notably,since no aortic valve obstruction or changes in its annulus diameterwere detected, the PSV parameter is likely reflecting the interactionbetween proximal aortic hemodynamics, and left ventricular systoliccontraction. Thus, PSV moderation by LA may represent attentuation ofboth aortic and LV remodeling.

LA also attenuated remodeling of the LV valve. AngII-induced thickeningof aortic and mitral valve leaflets was reduced by LA application inboth valves (p=0.06 in both valves, FIG. 22 left panels F-I).

The αSMA and TGFβ antigens were observed in aortic valve leaflet stromalcells in all AngII treated mice (FIG. 22 panels J-M′), decreasedproliferation of stromal cells in LA treated mice was demonstratedthrough Ki67 staining (p=0.26).

Thus, the present findings show that application of a leptin antagonistat the pivotal location on the proximal ascending aortic surfaceprevents rupture of thoracic aneurysms induced by systemic infusion ofAng II. Local inhibition of leptin activity reduces the degenerativeeffects of Ang II on the proximal aorta, which underlie aortic walldestabilization. Thus, moderation of Ang II induced aortic dilatationand attenuates left heart remodeling, presumably via theaorto-ventricular coupling.

These results highlight the role of leptin as a key mediator of Ang IIsignaling and indicate that leptin which underlie left ventricularhypertrophy also drives the formation of early aortic valve hyperplasticlesions, which may progress to aortic valve stenosis (AVS).

Example 3 The Role of Leptin in AVS Materials and Methods

Human AVS and normal arterial valve (AV) samples were collected foranalysis, including autopsy samples, freshly collected AVS specimensfrom patients undergoing aortic valve replacement surgery, and normalaortic valves from explanted hearts. Formalin fixed valve samples wereanalyzed by immunohistochemistry for leptin, leptin receptor, CD68 andαSMA. Fresh samples of AVS valves and normal aortic valves underwenttotal RNA extraction and analyzed by qPCR and Nanostring technique toassess leptin and leptin receptor mRNA levels. Retroperitoneal fat wasused as a positive control in both assays.

Results

Normal aortic valve leaflets lack leptin (Ob) antigen, and show very fewleptin receptor (ObR) positive cells (FIG. 23). Advanced AVS disease wascharacterized by extensive ossification and infiltration of inflammatorymacrophages in the non-calcified rim of cellular tissue (FIG. 24).Leptin was demonstrated mostly in two cell types, SMC-like elongatedcells, and macrophage-like round cells, and its prevalence wasproportional to the severity of AVS disease. In situ hybridizationanalysis performed on AVS samples demonstrated leptin mRNA expression,suggesting de novo synthesis (not shown) leptin and leptin receptor mRNAlevels were assessed by qPCR and Nanostrings hybridization, using totalRNA extracted from freshly collected AVS, AVS were compared to normal AVleaflets (FIG. 25), revealing increased leptin and leptin receptor mRNAin AVS samples.

To investigate the potential impact of AngII and leptin on human valvecells, in vitro analysis revealed that AngII-mediated proliferation ofhuman valve interstitial cells (VICs) is leptin mediated (leptin-inducedproliferation of VICs in FIG. 26). This suggests that leptin synthesizedin aortic valve leaflets by VICs and inflammatory macrophages may elicitVIC proliferation and subsequent ossification via a paracrine/autocrinepathways.

Example 4 Preparation of Sustained-Release Film

Poly lactic-co-glycolic acid (PLGA) films containing a leptin antagonist(e.g., a leptin antagonist described in U.S. Pat. No. 8,969,292) isproduced in a manner analogous to the described in Webber W L et al“Characterization of soluble, salt-loaded degradable PLGA films andtheir release of tetracycline” J Biomed Mater Res 1998, 41, 18-29.

Specifically, 1 g PLGA 6535 polymer (D,L-lactide:glycolide: 65:35,Mw=45,000-75,000 Da; Lakeshore. Biomaterials, Birmingham, Ala. USA) isdissolved in 10 mL MeCl₂ (Fisher Scientific, Loughborough, UK). Sodiumchloride (10 mg in 0.2 ml distilled water) and 25 μL ethylene glycol(Sigma-Aldrich, St. Louis, Mo., USA) are added to the PLGA solution andsonicated for 20 seconds. 1 mg of the leptin antagonist is suspended in2 mL of the PLGA solution, followed by casting on the flat surface of aTeflon mold to create a film comprising a leptin antagonist.

Example 5 Evaluation of Sustained-Release Properties of Film

A 2×2.5-mm patch of PLGA film of Example 4 is maintained in 5 mL sterilePBS at 37° C. for 28 days. Every 7 days the PBS medium is sampled beforebeing aspirated and replaced by 5 mL of fresh PBS. The medium samplesare analyzed by ELISA (Ray Biotech, Norcross, Ga., USA) for leptinantagonist levels. The in vitro measurement of leptin discharged fromthe slow-release PLGA film yields substantial release of leptinantagonist for at least two weeks.

Example 6 Preparation of Two-Component Gelling Sustained-release gelcomposition

A first component of the composition is an aqueous solution of modifiedcarboxymethyl cellulose with adipic dihydrazide (CMC-ADH). Dried andfinely ground sustained-release gel with leptin antagonist as describedin Example 1 is added to the CMC-ADH solution and vortexed to yield asuspension.

A second component of the composition is oxidized dextrane in DDW(DX-COH). A dye such as methylene blue (0.5%) is optionally added to theDX-COH solution to make the resulting gel more visible.

For use the two components are mixed together to form a viscous fluidthat is immediately administered by injection. In a short time, theviscous fluid gels.

Example 7 Covered Stents

A stent cover is fashioned from the PLGA film of Example 4.

The stent cover is used to cover the balloon expandable stents of aMULTI-LINK 8 LL coronary stent system, a MULTI-LINK ULTRA coronary stentsystem and a MULTI-LINK MINI VISION coronary stent system (all of AbbotLaboratories, Abbot Park, Ill., USA).

The graft (stent cover) portion of an Endurant II AAA Stent Graft System(Medtronic, Dublin, Ireland) is impregnated with a leptin antagonist. Inone example, impregnation is by immersion in a first component of thecomposition of Example 3, followed by contact with contact with thesecond component thereof.

The stent cover is used as an external cover for a self expandingWallFlex Stent (Boston Scientific Corporation, Natick, Mass., USA).

The covered stents are deployed in the usual way inside the lumen of ablood vessel of a living subject in need thereof. Once implanted, theleptin antagonist elutes from the stent cover through the blood vesselendothelium into the blood vessel to exert a desired pharmaceuticaleffect.

Example 8 Drug-Eluting Stent

An XIENCE Alpine coronary stent system (Abbot Laboratories, Abbot Park,Ill., USA) is prepared in the usual way, but impregnated with a leptinantagonist as an active pharmaceutical ingredient instead of Everolimus.

The resulting drug-eluting stent is deployed in the usual way inside thelumen of a blood vessel of a living subject in need thereof. Onceimplanted, the leptin antagonist elutes from the stent through the bloodvessel endothelium into the blood vessel to exert a desiredpharmaceutical effect.

Example 9 Bioresorbable Stent

A bioresorbable balloon-expandable stent is fashioned of bioresorbablepolylactide (PLA) comprising a leptin antagonist, substantially as doneto fashion a bioresorbable stent by Arterial Remodeling Technologies(Paris, France).

A bioresorbable balloon-expandable stent is fashioned of bioresorbablepoly (L-lactide) (PLLA) including a leptin antagonist, substantially asdone to fashion an Absorb GTI vascular scaffold stent.

The resulting bioresorbable stents are deployed in the usual way insidethe lumen of a blood vessel of a living subject in need thereof. Onceimplanted, the leptin antagonist elutes from the stents as these resorb,to pass through the blood vessel endothelium into the blood vessel toexert a desired pharmaceutical effect.

Example 10 Spike or Rod

An implantable spike or rod (bioresorbable or not) is made as known inthe art, for example as described above with reference to the stents andincludes leptin antagonist integrated into the material of the spike orrod, or adsorbed, absorbed or coated onto the spike or rod.

The resulting spike or rod is implanted, the leptin antagonist elutesfrom the spike or rod, to exert a desired pharmaceutical effect or theorgan.

Example 11 Injectable Gel

The two components of the composition of Example 3 are provided. Thecomponents are mixed together and implanted in the body of the subjectby injection into or onto an organ to form a gelled mass in or on theorgan.

Once implanted, the leptin antagonist elutes from the gelled mass, toexert desired pharmaceutical effect on the organ.

Example 12 Sheet

A sheet of the film of Example 1 is provided. The sheet is placedagainst the outer surface of an organ (e.g., aorta, for example,ascending aorta, aortic arch, descending aorta, abdominal aorta) andoptionally held in place by sutures and/or biological glue (e.g.,Evicel®

Once implanted, the leptin antagonist elutes from the film, to exert adesired pharmaceutical effect the organ.

Example 13 Treatment of AAA

A human subject is diagnosed with an abdominal aortic aneurysm (AAA).

A composition according to the teachings herein in the form of a longsheet such as described in Example 12 (a ribbon) comprising leptinantagonist is provided. The abdominal aorta of the subject is surgicallyaccessed from the outside (e.g., using keyhole surgery and thecomposition administered by winding the long sheet around the abdominalaorta to wrap the entire aneurysm as well as a portion of the aortaabove and below the aneurysm, and held in place with a biological glueand/or sutures. Optionally, a stent graft (e.g., an Endurant® II AAAStent Graft System (Medtronic, Dublin, Ireland)) is deployed in theaneurysm in the usual way, before or after administration of thecomposition. Subsequently, leptin antagonist from the composition passesthrough the tunica externa to provide a beneficial effect to thesubject,

Alternatively or additionally, an AAA stent graft is provided that issimilar to known AAA stent grafts (e.g., similar to an Endurant® II AAAStent Graft System (Medtronic, Dublin, Ireland)) where at least one of:the graft and/or anchoring stents are a composition of the teachingsherein and comprises a leptin antagonist; at least the anchoringportions of the graft and/or anchoring stents are impregnated with acomposition of the teachings herein that comprises a leptin antagonist;and at least the anchoring portions of the graft and/or anchoring stentsare coated with a composition of the teachings herein that comprises aleptin antagonist. The stent-graft is deployed in the usual way in theabdominal aneurysm, that is to say, where the anchoring stents areexpanded against healthy portions of tunica intima above and below theaneurysm. Subsequently, leptin antagonist from the composition passesthrough the tunica intima to provide a beneficial effect to the subject,for example, preventing or reducing the extent that the aneurysm spreadsto portions of tissue in proximity of the anchoring stents, therebypreventing loosening of the anchoring stents.

Alternatively or additionally, a drug-eluting balloon similar to theIn.Pact Admiral® DCB drug-coated balloon by Medtronic (Dublin, Ireland)that is coated with a composition comprising leptin antagonist accordingto the teachings herein is introduced through the femoral arteries andadvanced to areas of the abdominal and iliac arteries that are justabove and just below the aneurysm (for example, “landing zones” whereanchoring stents of a stent-graft would be deployed. The balloon isexpanded to contact the aorta and iliac walls, thereby administeringcomposition to the healthy tissue and preventing advancement of theaneurysm. In some such embodiments, the administration of leptinantagonist is repeated periodically, e.g., with a frequency that is lessthan once a month, less than once every two months and even less thanonce every 3 months. In some such embodiments, the administration of theleptin antagonist leads to stabilization of the wall tissue, halting theprocesses of aneurysm formation at the portions of the blood vesselsabove and below the aneurysm and at the portions of the blood vessel incontact with the deployed stent graft.

Example 14 Treatment of Thoracic Aortic Aneurysm

A human subject is diagnosed with an aneurysm in the thoracic aorta,including one or more of the ascending aorta, aortic arch and descendingaorta. It is known that segmental increased stiffness and aorticdilatation cause local aneurysm formation. These structural changesunderlie hemodynamic perturbation, which increases left ventricularafterload. This results in left ventricular remodeling, including leftventricular hypertrophy, and thickening of the aortic and mitral valveleaflets. Left ventricular hypertrophy may lead to heart failure, andaortic valve remodeling may progress to the full clinical presentationof aortic valve stenosis.

A composition according to the teachings herein the form of a patch suchas described in Example 12 comprising leptin antagonist is provided. Thethoracic aorta of the subject is surgically accessed from the outside(e.g., using thorascopy) and the composition administered by contactingthe outer surface of the affected portions of the aorta with the patch,and optionally holding the patch in place with a biological glue and/orsutures. Optionally, in cases of dilated ascending aorta beyond thesinuses of valsalva (preserved aortic valve annulus diameter) a drugeluting stent graft impregnated with leptin antagonist is deployed inthe aneurysm in the usual way, without oversizing.

Another shape of intra-vascular device that may be deployed within anaortic aneurysm is a tubular self-expandable biodegradable (ornon-degradable, like bare metal) leptin antagonist slow release mesh orstent. Such a device may self-expand upon deployment, and gently adhereto the luminal surface of the aortic aneurysm (applying minimal radialforce to the luminal surface). Leptin antagonist that is associatedwith, e.g., incorporated within the biodegradable or non-degradablestruts of the mesh or stent or covered or coated onto the mesh or stent,will access the aortic wall at the aneurysm by local diffusion. Yetanother shape of a device for proximity and local slow release of leptinantagonist at the luminal surface of aortic or peripheral aneurysm willbe a single wire (biodegradable or bare metal) possessing the memory ofspiral expansion within the aneurysm cavity. The leptin antagonistincorporated within this wire will diffuse into the arterial wall.Subsequently, leptin antagonist from the composition passes into thetissue to provide a beneficial effect to the subject, in someembodiments one or more of attenuate aneurysm progression, stabilize thevessel wall and prevent rupture or dissection of the aneurysm. In someembodiments of treatment of the ascending aorta, the administration ofthe leptin antagonist also leads to reducing the rate of development, orstopping the development and in some embodiments, reversing theremodeling of parts of the heart.

Example 15 Angioplasty

A human subject is diagnosed with arterial stenosis that is treatable byangioplasty.

A drug-eluting balloon similar to the In.Pact Admiral® DCB drug-coatedballoon by Medtronic (Dublin, Ireland) that is coated with a compositioncomprising leptin antagonist according to the teachings herein is usedin the usual way to perform the angioplasty procedure, for example, atsites of arterial bifurcations and in-stent stenoses. At least some ofthe composition according to the teachings herein that coats the balloonis administered to the surface of the treated blood vessel, therebyadministering a composition according to the teachings herein. In somesuch embodiments, the administration of leptin antagonist is repeatedperiodically, e.g., with a frequency that is less than once a month,less than once every two months and even less than once every 3 months,even when there is no express need for repeated angioplasty.Subsequently, leptin antagonist from the composition passes into and/orthrough the lesion and/or tunica intima to provide a beneficial effectto the subject.

Alternatively or additionally, at least one of:

a composition according to the teachings herein in the form of a stent;

a stent impregnated with a composition according to the teachingsherein;

a stent coated with a composition according to the teachings herein;

a composition according to the teachings herein in the form of a stentcover;

a stent cover impregnated with a composition according to the teachingsherein; and

a stent cover coated with a composition according to the teachingsherein;

is deployed in the usual way, e.g., during performance of an angioplastyprocedure, thereby administering a composition according to theteachings herein. Subsequently, leptin antagonist from the administeredcomposition passes into and/or through the lesion and/or tunica intimato provide a beneficial effect to the subject.

Example 16 Myocardial Infarction

Myocardial infarction causes left ventricular remodeling, leading toprogressive impairment of cardiac function. A human subject is diagnosedwith an acute myocardial infarction and is treated in the usual way, forexample coronary catheterization for primary revascularization andmyocardial salvage. A treating health-care professional identifies thatthe subject has an elevated risk of developing cardiac dysfunction.

A composition comprising leptin antagonist is administered to theascending aorta as described in the preceding examples using one or moreof a drug-eluting balloon, a stent, a covered stent and a stent graft.Subsequently, leptin antagonist from the administered composition passesinto and/or through the tunica intima of the aorta to provide abeneficial effect to the subject. In some embodiments, the beneficialeffect is prophylactic, preventing development of or reducing the rateof development of an thoracic aortic aneurysm, and/or remodeling of theheart (in particular the left ventricle and associated valves) and/or arecurring infarction. Without wishing to be held to any one theory, itis currently believed that such administration of a leptin antagonist inthe ascending aorta reduces angiotensin II synthesis in the leftventricle, thereby moderating the hypertrophy response to the ischemicinsult associated with the acute myocardial infarction suffered by thesubject.

Example 16—A Post Myocardial Ischemia (MI) Therapy

In order to minimize the extent of post MI left ventricular remodeling,a bolus of leptin antagonist in aqueous solution may be administeredinto the involved coronary artery. The following strategy may beexercised for acute MI patients: Patients who sustain acute MI are mostfrequently admitted through the catheterization lab, to undergo coronarycatheterization and primary PTCA (Percutaneous Transarterial CoronaryAngioplasty) for primary revascularization. Once blood-flow isre-established in the coronary artery involved, a bolus of leptinantagonist in an aqueous solution is to be infected into the coronaryartery through the catheter, afterwhich the catheter will be withdrawn.This new strategy should achieve local distribution of leptin antagonistwithin the left ventricular heart muscle cells (cardiomyocytes) thatwere exposed to the ischemic as well as reperfusion insult. Inhibitionof cardiomyocyte leptin activity is anticipated to mitigate leftventricular remodeling, and reduce the damage to left ventricularfunction.

Experimentally induced controlled myocardial ischemia may be achieved bytemporary balloon inflation within the proximal left anterior descending(LAD) coronary artery. Leptin antagonist aqueous solution may beinjected into the LAD after balloon deflation. Control group may receiveintracoronary bolus of saline injection.An intravascular injection of leptin antagonist may be provided into thetreated coronary artery after it has been reopened and blood flow to theischemic myocardium is restored, in order to prevent post MI leftventricular remodeling. Leptin antagonist may be administered byintravascular injection into a vascular territory that sustainedischemia and reperfusion injury. This injection is a localized injectionto a specific section of the left ventricle and does not constituted asystemic treatment.

Example 17 Myointimal Hyperplasia (MIH)

It is know that trauma to a blood vessel may lead to myointimalhyperplasia (MIH), where medial smooth muscle cells undergo uncontrolledproliferation that may lead to stenosis or restenosis of the bloodvessel in the area of the trauma. Such trauma include vascular injurycaused by expansion of a blood vessel during angioplasty, stentdeployment, stent-graft deployment, as a result of surgical anastomosisand associated suturing, clamping of blood vessels, and as a result ofblunt and/or penetrating vascular injury.

A health-care professional identifies that a subject has an elevatedrisk of developing myointimal hyperplasia due to some vascular trauma,administers a composition comprising leptin antagonist according to theteachings herein to the site of the trauma. The administered leptinantagonist reduces the rate or stops the uncontrolled proliferation ofcells, reducing the rate of development or preventing MIH.Administration includes the use of any of the compositions according tothe teachings herein, including localized administration of a leptinantagonist composition on the outer surface of the blood vessel at thesite of injury during surgery (e.g., application of a film of Example 4as a patch), intravascular administration using a drug eluting balloon,or by administration of a composition that impregnates or coats amedical device, or a composition that is in the shape of a medicaldevice by deploying the medical device. In some embodiments, specificsuitable medical devices include intracavitary devices such as a stentcover, a stent, a graft assembly, a ring, a suture and a prostheticcardiac valve as well as extraluminal devices such as sheets, all suchcomprising leptin antagonist according to the teachings herein.

Example 18 Treatment of Aneurysm

A human subject is diagnosed with a peripheral or venous aneurysm, e.g.,a visceral artery aneurysm, a cerebral aneurysm, especially a saccularor pseudo-fusiform aneurysm.

A composition according to the teachings herein associated with acovered stent is provided, e.g., one or more of coating the stent,coating the stent cover, impregnating the stent, impregnating the stentcover, constituting the stent and constituting the stent cover. Thecovered stent is deployed in the usual way, where the stent cover coversthe mouth of the stent. Subsequently, leptin antagonist from thecomposition passes into and/or through the tissue in proximity to theaneurysm to provide a beneficial effect to the subject.

Alternatively or additionally, a composition according to the teachingsherein is placed inside the cavity of the aneurysm through the mouththereof, e.g., as a fluid composition (e.g., Example 6) or as ananeurysm coil that is impregnated with leptin antagonist, coated withleptin antagonist or is made of a composition according to the teachingsherein. A person having ordinary skill in the art is able to implementcoating an aneurysm coil with an active pharmaceutical ingredient withreference to, for example, Cerectye® (Codman Neuro, a division ofDePuySynthes, part of Johnson & Johnson, New Brunswick, N.J., USA),Nexus® (Micro Therapeutics, Inc., Irvine, Calif., USA), and HydroCoil®,HydroSoft® (Terumo Corporation, Tokyo, Japan). Subsequently, leptinantagonist from the composition passes into the cavity of the aneurysm,and subsequently to affected tissue to provide a beneficial effect tothe subject.

Example 19 Treatment of Aneurysm

A human subject is diagnosed with an aneurysm, e.g., aortic aneurysms,which are typically related to a variety of diseases associated withangiotensin II hormonal activity. Also peripheral arterial aneurysms,which affect visceral, carotid, peripheral, and cerebral arteries, aswell as venous aneurysms, including pulmonary artery (which carriesvenous blood) may be diagnosed.

A leptin antagonist eluting stent or scaffold may be provided, e.g.,intravascular stent or scaffold device (which may or may not bebiodegradable) covered or coated with leptin antagonist, available forslow release into the vessel wall locally, thereby attenuating aorticaneurysm progression. A stent-graft destined for treatment of aorticaneurysm may be provided, covered or coated with leptin antagonistavailable for slow release into the vessel wall at the specific sites ofstent-graft attachment to non-dilated (normal) proximal and distalvessel (landing zones). In some embodiments, a stent-like prostheticheart valve for intravascular application, covered with leptinantagonist may be provided, to prevent local dilation of the hostingtissue ring.

Example 20 Treatment of Vascular Injury

A human subject may be exposed to localized vascular injury which mayoccur as a result of vascular surgery, local balloon angioplasty. Thismay cause local arterial narrowing due to smooth muscle cellproliferation, namely myointimal hyperplasia (MIH). This proliferativeresponse may also occur at arterio-venous anastomosis or graft-arterialanastomosis, and in leptin-induced inflammatory and cellularproliferative arterial disease (e.g., Takayasu disease). In order toprevent local arterial narrowing due to cellular proliferation at thesites of vascular injury, and circumstances as described above, anintravascular stent or scaffold device may be provided, (which may ormay not be biodegradable) which may be associated with, e.g., covered orcoated with leptin antagonist that is available for slow release intothe vessel wall.

Example 21 Treatment of Atherosclerotic Plaques

A human subject is diagnosed with atherosclerotic plaques. These lesionsfrequently undergo transformation from a stable plaque into an unstablerupture-prone lesion. An intravascular stent or scaffold device (whichmay or may not be biodegradable) covered with leptin antagonist that isavailable for slow release into the vessel wall may be provided, inorder to provide vessel stabilization. Deployment of such a stent orscaffold device may apply to any arterial site.

Example 22 Treatment of Left Heart Failure and Aortic Valve Disease

Patients who are diagnosed as hypertensive and hypercholestrolemic andexhibit initial dilation of the ascending aorta may be treated bypositioning an intravascular stent or scaffold device at the ascendingaorta. The stent or scaffold device may or may not be biodegradable. Thedevice, may be associated with, e.g., covered or coated with leptinantagonist that is available for slow release into the vessel wall, maycontrol left heart failure and attenuate the progression of aortic andmitral valve disease.

The intravascular stent or scaffold device carrying or covered withleptin antagonist, may be positioned intra-luminally at the ascendingaorta. Such a stent or scaffold device may attenuate local dilation andstiffening thereby prevent local hemodynamic perturbation, whichactivate the aorto-ventricular coupling. This coupling, when turned onpromotes the production and release of angiotensin II from leftventricular cells (cardiomyocytes). Angiotensin II drives intracellularsynthesis of leptin in cardiomyocytes and in aortic valve interstitialcells (VICs). Leptin synthesis in cardiomyocytes and VICs contributes toleft ventricular hypertrophy, and aortic/mitral valve thickening,respectively. Therefore, deactivation of the aorto-ventricular couplingwill control both left ventricular hypertrophy (heart failure) and alsoattenuate the progression of aortic/mitral valve disease.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention. Sectionheadings are used herein to ease understanding of the specification andshould not be construed as necessarily limiting.

TABLE 1 Sequence listing SEQ ID NOs Name Organism 1 leptin precursorHomo sapiens 2 leptin mRNA Homo sapiens 3 U.S. Pat. No. 7,307,142 seq1Artificial Sequence 4 U.S. Pat. No. 7,307,142 seq2 Artificial Sequence 5U.S. Pat. No. 7,307,142 seq3 Artificial Sequence 6 U.S. Pat. No.7,307,142 seq4 Artificial Sequence 7 U.S. Pat. No. 7,307,142 seq5Artificial Sequence 8 U.S. Pat. No. 7,307,142 seq6 Artificial Sequence 9U.S. Pat. No. 7,307,142 seq7 Artificial Sequence 10 U.S. Pat. No.7,307,142 seq8 Artificial Sequence 11 U.S. Pat. No. 7,307,142 seq9Artificial Sequence 12 U.S. Pat. No. 7,307,142 seq10 Artificial Sequence13 U.S. Pat. No. 7,307,142 seq11 Artificial Sequence 14 U.S. Pat. No.7,307,142 seq12 Artificial Sequence 15 U.S. Pat. No. 7,307,142 seq13Artificial Sequence 16 U.S. Pat. No. 7,307,142 seq14 Artificial Sequence17 U.S. Pat. No. 7,307,142 seq15 Artificial Sequence 18 U.S. Pat. No.7,307,142 seq16 Artificial Sequence 19 U.S. Pat. No. 7,307,142 seq17Artificial Sequence 20 U.S. Pat. No. 7,307,142 seq18 Artificial Sequence21 U.S. Pat. No. 7,307,142 seq19 Artificial Sequence 22 U.S. Pat. No.7,307,142 seq20 Artificial Sequence 23 U.S. Pat. No. 7,307,142 seq21Artificial Sequence 24 U.S. Pat. No. 7,307,142 seq22 Artificial Sequence25 U.S. Pat. No. 7,307,142 seq23 Artificial Sequence 26 U.S. Pat. No.7,307,142 seq24 Artificial Sequence 27 U.S. Pat. No. 7,307,142 seq25Artificial Sequence 28 U.S. Pat. No. 7,307,142 seq26 Artificial Sequence29 U.S. Pat. No. 7,307,142 seq27 Artificial Sequence 30 U.S. Pat. No.7,307,142 seq28 Artificial Sequence 31 U.S. Pat. No. 7,307,142 seq29Artificial Sequence 32 U.S. Pat. No. 7,307,142 seq30 Artificial Sequence33 U.S. Pat. No. 7,307,142 seq31 Artificial Sequence 34 U.S. Pat. No.7,307,142 seq32 Artificial Sequence 35 U.S. Pat. No. 7,307,142 seq33Artificial Sequence 36 U.S. Pat. No. 8,969,292 seq1 Artificial Sequence37 U.S. Pat. No. 8,969,292 seq2 Homo sapiens 38 U.S. Pat. No. 8,969,292seq3 Artificial Sequence 39 U.S. Pat. No. 8,969,292 seq4 ArtificialSequence 40 U.S. Pat. No. 8,969,292 seq5 Artificial Sequence 41 U.S.Pat. No. 8,969,292 seq6 Artificial Sequence 42 U.S. Pat. No. 8,969,292seq7 Artificial Sequence 43 U.S. Pat. No. 8,969,292 seq8 ArtificialSequence 44 U.S. Pat. No. 8,969,292 seq9 Artificial Sequence 45 U.S.Pat. No. 8,969.292 seq10 Artificial Sequence 46 U.S. Pat. No. 8,969,292seq11 Artificial Sequence 47 U.S. Pat. No. 8,969,292 seq12 ArtificialSequence 48 leptin precursor Rat Rattus norvegicus 49 US20070104708 seq1Mus musculus 50 US20070104708 seq2 Mus musculus 51 US20070104708 seq3Homo sapiens 52 US20070104708 seq4 Homo sapiens 53 US20070104708 seq5Homo sapiens 54 US20070104708 seq6 Homo sapiens 55 US20070104708 seq7Artificial Sequence 56 pcDNA3 Artificial Sequence 57 pcDNA3.1(+)Artificial Sequence 58 pcDNA3.1(−) Artificial Sequence 59 pGL3Artificial Sequence 60 pZeoSV2(+) Artificial Sequence 61 pSecTag2Artificial Sequence 62 pDisplay Artificial Sequence 63 pEF/myc/cytoArtificial Sequence 64 pCMV/myc/cyto Artificial Sequence 65 pCR3.1Artificial Sequence 66 pSinRep5 Artificial Sequence 67 pC1 ArtificialSequence 68 pMbac Artificial Sequence 69 pPbac Artificial Sequence 70pBK-RSV Artificial Sequence 71 pBK-CMV Artificial Sequence 72 pMT2Artificial Sequence

What is claimed is:
 1. A method of treating a cardiovascular disordercomprising: locally administering a leptin antagonist to acardiovascular tissue affected by said cardiovascular disorder fordown-regulating an expression or activity of leptin in saidcardiovascular tissue, wherein said leptin antagonist is a modifiedmammalian leptin peptide capable of binding a leptin receptor.
 2. Themethod of claim 1, wherein said leptin antagonist is selected from thegroup consisting of (a) a modified mammalian leptin peptide comprising amammalian leptin polypeptide in which the LDFI hydrophobic binding siteat the positions corresponding to positions 39-42 of the wild-type humanleptin (SEQ ID NO:1) is modified such that from two to four amino acidresidues of said hydrophobic binding site are substituted with differentamino acid residues such that the site becomes less hydrophobic; (b) themodified mammalian leptin polypeptide according to (a) in which theaspartic acid at the position corresponding to position 23 od thewild-type human leptin (D23) is substituted with an amino acid residueselected from the group consisting of glycine, alanine, leucine, lysine,arginine, phenylalanine, tryptophan and histidine, or in which thethreonine at the position corresponding to position 12 of the wild-typehuman leptin (T12) is substituted with a different amino acid residuethat is hydrophobic; and (c) a pharmaceutically acceptable salt of (a)or (b).
 3. The method of claim 1, wherein said leptin antagonist whereinsaid modified mammalian leptin peptide is of an amino acid sequenceselected from SEQ ID NOs: 3-10, 36 and 38
 4. The method of claim 1,wherein said cardiovascular disorder is characterized by tissueremodeling.
 5. The method of claim 1, wherein said cardiovasculardisorder is selected from the group consisting of vascular aneurysm,aortic aneurysm, atherosclerotic plaques, left ventricular remodeling,myocardial infarction, myointimal hyperplasia, vascular injury, leftheart failure and aortic or mitral valve disease.
 6. The method of claim1, wherein said cardiovascular disorder is selected from the groupconsisting of cardiovascular disease; remodeling of stableathersclerotic plaque into an unstable lesion; ascending aortic aneurysmassociated with hypertension, dyslipidemia, hypercholesterolemia,obesity, diabetes mellitus or bicuspid aortic valve; Takayasu disease;rheumatoid arteritis; giant cell arteritis; inflammatory aorticaneurysm; thoracic aortic aneurysm; abdominal aortic aneurysm; aorticaneurysm and/or pulmonary artery aneurysm in Marfan syndrome; aorticdissection in an aortic or peripheral large artery; angiogenesis; andarteriovenous malformation.
 7. The method of claim 1, wherein saidcardiovascular tissue is arterial or venous wall tissue.
 8. The methodof claim 1, wherein said cardiovascular tissue is aortic and/or mitralheart valve leaflet tissue.
 9. The method of claim 1, wherein saidcardiovascular tissue is an inner (luminal) surface of an aorta or anouter wall of an aorta, within or on the surface of a vascular organ.10. The method of claim 1, wherein said locally administrating iseffected by means of a carrier configured for localized release of saidleptin antagonist.
 11. The method of claim 1, wherein said locallyadministrating is effected via direct injection.
 12. The method of claim10, wherein said carrier is a solid, gel or liquid carrier.
 13. Themethod of claim 10, wherein said carrier comprises a biodegradablesupport.
 14. The method of claim 10, wherein said carrier is configuredas a film.
 15. The method of claim 10, wherein said carrier is a deviceselected from the group consisting of a gel, mesh, a balloon and avascular graft.
 16. The method of claim 10, wherein said carrier is adepot-forming injectable composition.
 17. The method of claim 10,wherein said carrier is a scaffold configured for slow release and saidcardiovascular tissue is the luminal surface of an aorta, and whereinsaid scaffold for slow release is applied to said luminal surface of theaorta via arterial catheterization.
 18. The method of claim 17, whereinsaid scaffold is biodegradable.
 19. The method of claim 1, wherein saidlocally administrating is effected intracavitarily.